DEPARTMENT OF BIOTECHNOLOGY

B.TECH BIOTECHNOLOGY
BATCH (2019 – 2023)II YR IV SEM
BT 8402 MOLECULAR BIOLOGY – QUESTION BANK

Prepared by

G.GOMATHI SANKAR, M.TECH

ASSISTANT PROFESSOR

DEPT. OF BIOTECHNOLOGY

G. Gomathi
S.N TOPIC REFER PAGE
O ENCE NO
UNIT I – CHEMISTRY OF NUCLEIC ACIDS
1 Introduction to nucleic acids TB1 79
2 Nucleic acids as genetic material TB1 79
3 Structure and physicochemical properties of elements in DNA and RNA TB1 80-82
4 Biological significance of differences in DNA and RNA TB1 84-92
5 Primary structure of DNA: Chemical and structural qualities of 3’,5’- TB1 94-96
Phosphodiester bond
6 Secondary Structure of DNA: Watson & Crick model TB1 97-100
7 Chargaff’s rule TB1 109
8 X–ray diffraction analysis of DNA, Forces stabilizes DNA structure TB1 110-112
9 Conformational variants of double helical DNA, Hogsteen base pairing TB1 114
10 Triple helix, Quadruple helix, Reversible denaturation and hyperchromic effect TB1 112-114
11 Tertiary structure of DNA: DNA supercoiling. TB1 148
UNIT II – DNA REPLICATION & REPAIR
1 Overview of Central dogma TB1 208
2 Organization of prokaryotic and eukaryotic chromosomes DNA replication: TB1 209
Meselson & Stahl experiment, bi–directional DNA replication
3 Okazaki fragments TB1 210
4 Proteomics of DNA replication, Fidelity of DNA replication TB1 224-231
5 Inhibitors of DNA replication TB1 232-235
6 Overview of differences in prokaryotic and eukaryotic DNA replication TB1 271-273
7 Telomere replication in eukaryotes TB1 239-246
8 D-loop and rolling circle mode of replication TB1 255-258
9 Mutagens, DNA mutations and their mechanism, various types of repair TB1 293-305
Mechanisms
UNIT III – TRANSCRIPTION
1 Structure and function of mRNA, rRNA and tRNA TB1 117
2 Characteristics of promoter and enhancer sequences TB1 316
3 RNA synthesis: Initiation, elongation and termination of RNA synthesis, TB1 317-329
Proteins of RNA synthesis
4 Fidelity of RNA synthesis TB1 342
5 Inhibitors of transcription, Differences in prokaryotic and eukaryotic TB1 343-349
Transcription
6 RNA processing: 5’-Capping, Splicing-Alternative splicing, Poly ‘A’ tail TB1 352-363
addition and base modification
UNIT IV – TRANSLATION
1 Introduction to Genetic code: Elucidation of genetic code, Codon degeneracy TB1 367-378
2 Wobble hypothesis and its importance TB1 379
3 Prokaryotic and eukaryotic ribosomes TB1 439-447
4 Steps in translation: Initiation, Elongation and termination of protein synthesis TB1 425-435
5 Inhibitors of protein synthesis. Post- translational modifications and its TB1 441-445
Importance
UNIT V – REGULATION OF GENE EXPRESSION
1 Organization of genes in prokaryotic and eukaryotic chromosomes, TB1 453-454,
Hierarchical levels of gene regulation 502-510
2 Prokaryotic gene regulation –lac and trp operon
G.TB1 456-462,
Gomathi
479 – 480
3 Regulation of gene expression with reference to λ phage life cycle TB1 598 – 613
TB1 - Friefelder, David. “ Molecular Biology.” Narosa Publications, 1999

BT8402 MOLECULAR BIOLOGY LT P C 3003

OBJECTIVES:
 Familiarize students with the cell and molecular biology of both Prokaryotes and
Eukaryotes. This will be needed for any project work in modern biotechnology.
 By doing this course students will acquire basic fundamental knowledge and explore
skills in molecular biology and become aware of the complexity and harmony of the
cells.
 This course will emphasize the molecular mechanism of DNA replication, repair,
transcription, protein synthesis and gene regulation in various organisms.

UNIT I CHEMISTRY OF NUCLEIC ACIDS 9

Introduction to nucleic acids: Nucleic acids as genetic material, Structure and

physicochemical properties of elements in DNA and RNA, Biological significance of
differences in DNA and RNA. Primary structure of DNA: Chemical and structural qualities
of 3’,5’-Phosphodiester bond. Secondary Structure of DNA: Watson & Crick model,
Chargaff’s rule, X–ray diffraction analysis of DNA, Forces stabilizes DNA structure,
Conformational variants of double helical DNA, Hogsteen base pairing, Triple helix,
Quadruple helix, Reversible denaturation and hyperchromic effect. Tertiary structure of
DNA: DNA supercoiling.

UNIT II DNA REPLICATION & REPAIR 9

Overview of Central dogma. Organization of prokaryotic and eukaryotic chromosomes. DNA

replication: Meselson & Stahl experiment, bi–directional DNA replication, Okazaki
fragments, Proteomics of DNA replication, Fidelity of DNA replication, Inhibitors of DNA
replication, Overview of differences in prokaryotic and eukaryotic DNA replication,
Telomere replication in eukaryotes. D-loop and rolling circle mode of replication. Mutagens,
DNA mutations and their mechanism, various types of repair mechanisms.

UNIT III TRANSCRIPTION 9

Structure and function of mRNA, rRNA and tRNA. Characteristics of promoter and enhancer
sequences. RNA synthesis: Initiation, elongation and termination of RNA synthesis, Proteins
of RNA synthesis, Fidelity of RNA synthesis, Inhibitors of transcription, Differences in
prokaryotic and eukaryotic transcription. Basic concepts in RNA world: Ribozymes, RNA
processing: 5’- Capping, Splicing-Alternative splicing, Poly ‘A’ tail addition and base
modification.

UNIT IV TRANSLATION 9
Introduction to Genetic code: Elucidation of genetic code, Codon degeneracy, Wobble
hypothesis and its importance, Prokaryotic and eukaryotic ribosomes. Steps in translation:
Initiation, Elongation and termination of protein synthesis. Inhibitors of protein synthesis.
Post- translational modifications and its importance.
G. Gomathi
UNIT V REGULATION OF GENE EXPRESSION 9

Organization of genes in prokaryotic and eukaryotic chromosomes, Hierarchical levels of

gene regulation, Prokaryotic gene regulation –lac and trp operon, Regulation of gene
expression with reference to λ phage life cycle.

TOTAL : 45 PERIODS

OUTCOMES:
By the end of this course, students should be able to:
 Describe the basic structure and biochemistry of nucleic acids and proteins
and discriminate between them;
 Identify the principles of DNA replication, transcription and translation and
explain how they relate to each other.
 Discuss clearly about gene organization and mechanisms of control the
gene expression in various organisms.
 Articulate applications of molecular biology in the modern world.

TEXT BOOKS:
1. Friefelder, David. “ Molecular Biology.” Narosa Publications, 1999.
2. Weaver, Robert F. “ Molecular Biology” 2nd Edition, Tata McGraw-Hill,2003.
3. Karp, Gerald “ Cell and Molecular Biology : Concepts and Experiments” 4th Edition, John
Wiley, 2005.
4. Friefelder, David and George M. Malacinski “Essentials of Molecular Biology” 2nd
Edition, Panima Publishing, 1993.
5. Lewin’s GENES XI, Published by Jones & Bartlett Learning; 11 edition (January 15, 2013).
REFERENCES:
1. Tropp, Burton E. “ Molecular Biology : Genes to Proteins”. 3rd Edition. Jones and
Bartlett, 2008.
2. Glick , B.R. and J.J. Pasternak. “Molecular Biotechnology : Principles and Applications of
Recombinant DNA” 4th Edition. ASM, 2010.

G. Gomathi
 TWO MARKS

UNIT I: CHEMISTRY OF NUCLEIC ACIDS

1. Why do the absorption increases upon denaturation of dsDNA (May 2019)

The two strands of DNA are bound together mainly by the stacking interactions, hydrogen bonds
and hydrophobic effect between the complementary bases. The hydrogen bond limits the resonance
of the aromatic ring so the absorbance of the sample is limited as well. When the DNA double
helix is treated with denatured agents, the interaction force holding the double helical structure is
disrupted. The double helix then separates into two single strands which are in the random coiled
conformation. At this time, the base-base interaction will be reduced, increasing the UV absorbance
of DNA solution because many bases are in free form and do not form hydrogen bonds with
complementary bases. As a result, the absorbance for single-stranded DNA will be 37% higher than
that for double stranded DNA at the same concentration.
2. Explain Chargaff’s rule. (C212.1) (PO1,2,3,4) (May 2019)

Chargaff's rules states that DNA from any cell of all organisms should have a 1:1
ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount
of guanine is equal to cytosine and the amount of adenine is equal to thymine.

3. What is okazaki fragment? (C212.1) (PO1,2,3,4) (May 2019)

Okazaki fragments are short, newly synthesized DNA fragments that are formed on
the lagging template strand during DNA replication. They are complementary to the lagging
template strand, together forming short double-stranded DNA sections. Okazaki fragments
are between 1,000 to 2,000 nucleotides long in Escherichia coli and are between 100 to 200
nucleotides long in eukaryotes. They are separated by ~10-nucleotide RNA primers and are
unligated until RNA primers are removed, followed by enzyme ligase connecting (ligating)
the two Okazaki fragments into one continuous newly synthesized complementary strand

4. Define nucleic acid. (C212.1) (PO1,2,3,4)

Nucleic acids are the polynucleotides having high molecular weight. The monomeric
unit of which is nucleotide. Nucleic acids are biopolymers, or large biomolecules, essential
for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic
acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Each
nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous
base. If the sugar isdeoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is
RNA. When all three components are combined, they form a nucleic acid

5. Define bacterial transformation. (C212.1) (PO1,2,3,4)

Bacterial transformation the exchange of genetic material between strains of bacteria

by the transfer of a fragment of naked DNA from a donor cell to a recipient cell, followed by
recombination in the recipient chromosome.
G. Gomathi
6. What are the properties of genetic material? (C212.1) (PO1,2,3,4)
 Stores genetic information, Physical and Chemical stability (The double stranded
protects the DNA from chemical attack) Able to undergo mutation Stored information
accessible to progeny

7. Explain cantenation. (C212.1) (PO1,2,3,4)

Catenation is the ability of a chemical element to form a long chain-like structure via
a series of covalent bonds. Catenation occurs most readily in carbon, which forms covalent
bonds with other carbon atoms

8. What are Satellite DNA? (C212.1) (PO1,2,3,4)

Satellite DNA consists of very large arrays of tandemly repeating, non-coding DNA.
Satellite DNA is the main component of functional centromeres, and form the main structural
constituent of heterochromatin.
The name "satellite DNA" refers to how repetitions of a short DNA sequence tend to produce
a different frequency of the nucleotides adenine, cytosine, guanine and thymine, and thus
have a different density from bulk DNA - such that they form a second or 'satellite' band
when genomic DNA is separated on a density gradient

9. Differentiate prokaryotic and eukaryotic promoters. (C212.1) (PO1,2,3,4)

Prokaryotic promoters
In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions
upstream from the transcription start site. The sequence at -10 is called the Pribnow box, or
the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is
absolutely essential to start transcription in prokaryotes. The other sequence at -35 (the -35
element) usually consists of the six nucleotides TTGACA. Its presence allows a very high
transcription rate.
Eukaryotic promoters
Eukaryotic promoters are extremely diverse and are difficult to characterize. They typically
lie upstream of the gene and can have regulatory elements several kilobases away from the
transcriptional start site. In eukaryotes, the transcriptional complex can cause the DNA to
bend back on itself, which allows for placement of regulatory sequences far from the actual
site of transcription. Many eukaryotic promoters, contain a TATA box (sequence TATAAA),
which in turn binds a TATA binding protein which assists in the formation of the RNA
polymerase transcriptional complex. The TATA box typically lies very close to the
transcriptional start site (often within 50 bases).

8.What are the three enzymatic activities for DNA polymerase I? (C212.1) (PO1,2,3,4)

DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA
replication and is exclusively found in prokaryotes. It is composed of 928 amino acids, and is
an example of a processive enzyme - it can sequentially catalyze multiple polymerisations.
Discovered by Arthur Kornberg in 1956,[1] it was the first known DNA polymerase (and,
indeed, the first known of any kind of polymerase). It was initially characterized in E. coli,
although it is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that
encodes Pol I is known as polA.
G. Gomathi
9.Describe the biological significance of nucleic acids. (C212.1) (PO1,2,3,4)
 Nucleic acids are large molecules that carry tons of small details: all the genetic
information. Nucleic acids are found in every living thing — plants, animals, bacteria,
viruses, fungi — that uses and converts energy. Every single living thing has something in
common. People, animals, plants, and more all are connected by genetic material. Every
living thing may look different and act different, but deep down — way deep down in the
nucleus of cells — living things contain the same chemical “ingredients” making up very
similar genetic material. There are two types of nucleic acids: DNA (which stands for
deoxyribonucleic acid) and RNA (which stands for ribonucleic acid). Nucleic acids are made
up of strands of nucleotides, which are made up of a base containing nitrogen (called a
nitrogenous base), a sugar that contains five- carbon molecules, and a phosphoric acid.

11. Describe the properties of phosphodiester bond (C212.1) (PO1,2,3,4)

In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon
atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and
ribose in RNA. Strong covalentbonds form between the phosphate group and two 5-carbon
ring carbohydrates (pentoses) over two ester bonds.

12.Explain secondary structure of DNA. (C212.1) (PO1,2,3,4)

Secondary structure is the set of interactions between bases, i.e., which parts of
strands are bound to each other. In DNA double helix, the two strands of DNA are held
together by hydrogen bonds. The nucleotides on one strand base pairs with the nucleotide on
the other strand.

13.List out the forces that stabilize the structure of DNA(C212.1) (PO1,2,3,4)

The nitrogenous bases are positioned inside the helixstructure like "rungs on a
ladder," due to the hydrophobic effect, and stabilized by hydrogen bonding. The two strands
run in opposite directions to form the double helix. The strands are held together by hydrogen
bonds and hydrophobic interactions.

14.Define secondary structure of DNA. (C212.1) (PO1,2,3,4)

Secondary structure is the set of interactions between bases, i.e., which parts of
strands are bound to each other. In DNA double helix, the two strands of DNA are held
together by hydrogen bonds. The nucleotides on one strand base pairs with the nucleotide on
the other strand.

15. Define phosphor diester bond. (C212.1) (PO1,2,3,4)

1.
G. Gomathi
A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric
acid react with hydroxyl groups on other molecules to form two ester bonds. An example is
found in the linking of twopentose (5 carbon sugar) rings to a phosphate group by
strong, covalent ester bonds. Each ester bond is formed by a condensation reaction in which
water is lost. This bond is a key structural feature of the backbone of DNA and RNA and
links the 3’ carbon of one nucleotide to the 5’ carbon of another to produce the strands of
DNA and RNA. In phosphodiester formation, two hydroxyl (OH) groups on the
phosphate molecule bind to the 3’ and 5’ carbons on two independent pentose sugars. These
are two condensation reactions, so two molecules of water are produced. The phosphate is
then bonded to the sugars by two ester bonds, hence the nomenclature of phosphodiester
bond. This reaction is catalysed by ligases, such as DNA ligase during DNA replication.

16. Define hogsteen base pairing. (C212.1) (PO1,2,3,4)

A Hoogsteen base pair is a variation of base-pairing in nucleic acids such as the A. •

T pair. In this manner, two nucleobases, one on each strand, can be held together by
hydrogen bonds in the major groove.

17. Define a triple helix DNA. (C212.1) (PO1,2,3,4)

DNA can form multi-stranded helices through either folding of one of the two strands
or association of two, three, or four strands of DNA. Triple-helical nucleic acids were first
described in 1957 by Felsenfeld and Rich, who demonstrated that polyuridylic acid and
polyadenylic acids strands in a 2:1 ratio were capable of forming a stable complex. In 1986, it
was demonstrated that a short (15-mer) mixed-sequence triplex-forming oligonucleotide
(TFO) formed a stable specific triple helical DNA complex . The third strand of DNA in the
triplex structure (i.e.the TFO) follows a path through the major groove of the duplex DNA.
The specificity and stability of the triplex structure is afforded via Hoogsteen hydrogen bonds
, which are different from those formed in classical Watson-Crick base pairing in duplex
DNA. Because purines contain potential hydrogen bonds with incoming third strand bases,
the binding of the third strand is to the purine-rich strand of the DNA duplex.

18. Explain the quadruple structure of DNA. (C212.1) (PO1,2,3,4)

The structures are called G-quadruplexes, because they form in regions of DNA that
are full of guanine, one of the DNA molecule’s four building blocks. The others are adenine,
cytosine and thymine. A hydrogen bond is responsible for holding the four guanines together.
The four stranded DNA usually presents itself right before cell division.
G. Gomathi
19. Explain the hyperchromic effect of DNA. (C212.1) (PO1,2,3,4)
 DNA's hyperchromic effect means that ssDNA absorbs more UV than does dsDNA).
An insect, which can see in the UV range,* would see the hyperchromic effect something like
that shown to the right. At first the DNA solution is only a little violet. If it is boiled and then
slowly cooled, it ends up a little more violet than it started, but if it is rapidly cooled it
becomes most violet. The reason that this happens is that in dsDNA the pi-electrons in the
aromatic rings are more constrained because the H-bonded rings are in sandwich layers -
overlapping with each other. But if the H-bonds are "boiled" away, the sandwich no longer
exists and the pi-electrons are more free to move into different energy levels and thus able to

absorb more UV energy.

20. Define a super coiled DNA. (C212.1) (PO1,2,3,4)

A double helix (as of DNA) that has undergone additional twisting in the same
direction as or in the opposite direction from the turns in the original helix. The term
"supercoiling" means literally the coiling of a coil. A telephone cord for example, is typically
a coiled wire. The twisted path often taken by that wire as it goes from the base of the
phone to the receiver
generally describes a supercoil. DNA is coiled in the form of a double helix. Let us define an
axis about which both strands of the DNA coil. A bending or twisting of that axis upon itself
is referred to as DNA supercoiling. DNA supercoiling is generally a manifestation of
structural strain. Conversely, if there is no net bending of the DNA axis upon itself, the DNA
is said to be in a relaxed state. It is probably apparent that DNA compaction must involve
some form of supercoiling. Perhaps less apparent is the fact that replicating or transcribing
DNA also must induce some degree of supercoiling.

21. What is the basic difference between B and Z type of DNA? (C212.1) (PO1,2,3,4)
B Type:
In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each
strand looks like a helix. The two strands form a "double helix" structure, which was first
discovered by James D. Watson and Francis Crick in 1953. In this structure, also known as
the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring
base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. The intertwined strands make
two grooves of different widths, referred to as the major groove and the minor groove,
which may facilitate binding with specific proteins.
Z Type:
Another DNA structure is called the Z form, because its bases seem to zigzag. Z DNA is left-
handed. One turn spans 4.6 nm, comprising 12 base pairs. The DNA molecule G. Gomathi
with
alternating G-C sequences in alcohol or high salt solution tends to have such structure.
22. Relate hyperchromocity and denaturation of DNA(C212.1) (PO1,2,3,4)

Hyperchromicity is the increase of absorbance (optical density) of a material. The

most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is
denatured. The UV absorption is increased when the two single DNA strands are being
separated, either by heat or by addition of denaturant or by increasing the pH level. The
opposite, a decrease of absorbance is called hypochromicity. Heat denaturation of DNA, also
called melting, causes the double helix structure to unwind to form single stranded DNA.
When DNA in solution is heated above its melting temperature (usually more than 80 °C), the
double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked
and can thus absorb more light.

23. Mention two differences between prokaryotic DNA polymerase I and II(C212.1) (PO1,2,3,4)

DNA polymerase I DNA polymerase II

1 Composed of 928 amino acids Composed of 783 amino acids
2 Belongs to polymerase family A Belongs to polymerase family B
3 Responsible for DNA repair and removing Responsible for proofreading, fidelity, and
RNA primers processivity of newly formed DNA

24. What is a molecular 'chaperone'? (C212.1) (PO1,2,3,4)

Most biological structures assemble by themselves into larger structures; some (e.g.,
the icosahedral DNA bacteriophage P22) require help from an additional molecule that is
not found in the final structure.

25. What properties of a protein ensure that it is localized to a particular part of a cell? (C212.1)
(PO1,2,3,4)

Nuclear, cytoplasmic, and extracellular proteins each have characteristic surfaces

(cytoplasmic proteins have a balance of acidic and basic residues, extracellular proteins a
slight excess of acidic residues, and nuclear proteins a pronounced excess of basic
residues); then, we might imagine that newly-made proteins diffuse throughout the cell, to
bind at a specific location. Individual proteins also have short peptide sequences (e.g.,
nuclear localization signals) that target the whole protein to a specific subcellular
compartment (e.g., the nucleus).

26. Why are nuclei often round in shape? (C212.1) (PO1,2,3,4)

Surface tension is a major determinant. The nucleus has a fluid system that is
immiscible with its surroundings. Its surface tends towards the spherical minimum, and it
is immersed in a medium that transmits on all sides a uniform fluid (hydrostatic) pressure;
therefore, a nucleus is spherical.

27. Outline the general structure of the nucleosome. What is the evidence for this structure? (C212.1)
(PO1,2,3,4)

Structure: 146 bp of duplex DNA wrapped around a histone octamer (two copies of H3,
H4, H2A, H2B) in 1.65 turns of a flat, left-handed superhelix. Evidence: G. Gomathi
2 copies of
each core histone (and ~1 of H1) per ~200 bp DNA, the 'beads-on-a- string' structure
seen by electron microscopy, nuclease digestion of linker DNA to give a nucleosomal
 ladder (repeat 180-260 bp) following by trimming of the linker to give the core particle
with 146 bp DNA, the structure of the core particle determined by X-ray
crystallography.

28. How can the lengths of DNA in the nucleosome, and between nucleosomes, be determined? (C212.1)
(PO1,2,3,4)

By progressive digestion of chromatin with nucleases (e.g., micrococcal nuclease),

purification of DNA, and sizing the resulting fragments by gel electrophoresis. The
distance between 'rungs' in the resulting nucleosomal 'ladder' gives the repeat length,
while the length of the most resistant fragments gives the length of DNA in the
nucleosome. The length of the linker can be obtained by subtraction.

29. Describe the evidence for and against the existence in vivo of a 'solenoid'. (C212.1) (PO1,2,3,4)

For: electron microscopy of chromatin fibres in buffers of low ionic strength reveals
helical solenoids with six to eight nucleosomes/turn and an 11 nm pitch.
Against: solenoids are not seen at a physiological salt concentration, or in
electron micrographs of rapidly-frozen whole cells.

30. What is the evidence that the chromatin fiber is organized into loops in the interphase nucleus?
(C212.1) (PO1,2,3,4)

it seems inconceivable that long DNA molecules are packed randomly like spaghetti,
(ii) direct observation of lampbrush chromosomes (loops visible in unfixed material,
but they might form when nuclei are dispersed), (iii) the demonstration of
supercoiling in 'nucleoids' (but loops might be created artifactually during reparation),
(iv) the rate at which nucleases release chromatin from interphase nuclei (but isolated
in hypotonic buffers) or permeabilized cells (isolated in isotonic buffers). Go onto
discuss the analysis of residual fragments, and how the results suggest that
attachments (and so loops) change continually.

G. Gomathi
31) Differentiate primary and secondary structure of DNA(C212.1) (PO1,2,3,4)

Primary structure:
Sequence of nucleotide chains. It is in these channels where the genetic information, and
because the skeleton is the same for all the difference in the information lies in the
different sequence of nitrogenous bases. This sequence has a code, which determines an
information or otherwise, as the order of the bases.
Secondary structure:
It is a double helix structure. Can explain the storage of genetic information and the
mechanism of DNA replication. It was postulated by Watson and Crick, based on X-ray
diffraction that Franklin and Wilkins had been made, and the equivalence of bases
Chargaff, whereby the sum of adenines more guanines is equal to the sum of thymines
more cytokines.
It is a double strand, right-handed or left-handed, depending on the DNA. Both chains are
complementary, as adenine and guanine in a chain are joined, respectively, thymine and
cytosine on the other. Both chains are antiparallel, then the 3 'end of one faces the 5' end
of the counterpart.
32) What are nucleic acids? What is the historical origin of their name? (C212.1)
(PO1,2,3,4)

DNA and RNA, the nucleic acids, are the molecules responsible for the hereditary
information that controls the protein synthesis in living organisms. The name “nucleic”
derives from the fact that they were discovered (by the Swiss biochemist Friedrich Miescher,
in 1869) within the cell nucleus. At that time, it was not known that those substances
contained hereditary information.

33) What units make up nucleic acids? What are the chemical compounds that make
up those units? (C212.1) (PO1,2,3,4)

Nucleic acids are formed by sequences of nucleotides.

Nucleotides are composed of one molecule of sugar (deoxyribose in DNA and ribose in
RNA) bound to one molecule of phosphate and to one nitrogenous base (adenine, uracil,
cytosine or guanine, in RNA; and adenine, thymine, cytosine and guanine, in DNA).

34) What are pentoses? To what organic group do pentoses belong? Are nucleotides
formed of only one type of pentose? (C212.1) (PO1,2,3,4)

Pentoses are carbohydrates made of five carbons. Deoxyribose is the pentose that composes
DNA nucleotides and ribose is the pentose contained in RNA nucleotides. G. Gomathi
35) Into which two groups can the nitrogenous bases that form DNA and RNA be
classified? What is the criterion used in that classification? (C212.1) (PO1,2,3,4)

The nitrogenous bases that form DNA and RNA are classified as pyrimidine and purine
bases.

Through the analysis of the structural formulae of those nitrogenous bases, it is possible to
see that three of them, cytosine, thymine and uracil, have only one nitrogenized carbon ring.
The others, adenine and guanine, have two nitrogenized bound carbon rings.

36) What is the difference between DNA and RNA from the point of view of the
nitrogenous bases that are present in their nucleotides? (C212.1) (PO1,2,3,4)

In DNA, nucleotides can be made up of adenine (A), thymine (T), cytosine (C) or guanine
(G). In RNA, nucleotides can also contain adenine (A), cytosine (C) or guanine (G); however,
instead of thymine (T), they contain uracil (U).

37) What parts of nucleotides bind to form nucleic acids? What is meant by the 5’ and
3’ extremities of nucleic acids? (C212.1) (PO1,2,3,4)

The phosphate group of one nucleotide binds to the pentose of the other nucleotide and so on
to make the polynucleotide chain.

Each extremity of a DNA or RNA chain can be distinguished from the other extremity by its
terminal chemical entity. The phosphate-ended extremity is called a 5’-extremity and the
pentose-ended extremity is called a 3’-extremity. Therefore, DNA or RNA chains can have a
5’-3’ or 3’-5’ direction. These directions are important for several biological functions of
DNA and RNA, since some reactions specifically occur in one direction or the other.

38) Bacteria are prokaryotic cells, meaning that they do not have a membrane-
enclosed nucleus. Eukaryotes have cells with am enclosed nucleus. Where in these
types of cells can DNA be found? (C212.1) (PO1,2,3,4)

In eukaryotic cells, DNA is found within the cell nucleus. In prokaryotic cells, DNA is found
dispersed in the cytosol, the fluid space inside the cell.

Other DNA molecules can also be found within mitochondria and chloroplasts, specialized
organelles of eukaryotic cells.

39) Who were James Watson, Francis Crick and Maurice Wilkins? (C212.1) (PO1,2,3,4)
G. Gomathi
 Watson (American), Crick (British) and Wilkins (New Zealander) were responsible for the
discovery of the molecular structure of DNA, the double helix made of two polynucleotide
chains paired by their nitrogenous bases. They won the Nobel Prize in medicine in 1962 for
the discovery.

40) According to the Watson-Crick model, how many polynucleotide chains does a
DNA molecule have? (C212.1) (PO1,2,3,4)

A DNA molecule is formed by two polynucleotide chains bound in antiparallel mode (5’-3’
to 3’-5’) and which form a helix structure.

41) What is the rule for the pairing of nitrogenous bases within the DNA
molecule? What about in RNA molecules? Is this last question relevant? (C212.1)
(PO1,2,3,4)

The rule for the pairing of the nitrogenous bases of the polynucleotide chains that form DNA
molecules is that pyrimidine base binds to purine base, under the condition that thymine (T)
binds to adenine (A) and cytosine (C) binds to guanine (G).

In RNA, there is no binding between nitrogenous bases. That is because RNA is formed of
only one polynucleotide chain, as opposed by DNA, which is formed of two chains. It is
therefore not correct to ask questions about base pairing in RNA.

42) What is the numeric relationship between pyrimidine and purine bases in DNA
molecules? Is this valid for RNA molecules? (C212.1) (PO1,2,3,4)

DNA molecules are made of two bound polynucleotide chains that form a helix structure (the
double helix). The binding of the two chains occurs between their nitrogenous bases and
always obeys the following rules: adenine (A), a purine base, binds with thymine (T), a
pyrimidine base; and guanine (G), a purine base, binds to cytosine (C), a pyrimidine base.
Therefore, in one molecule of DNA, there will be the same number of adenine (A) and
thymine (T) bases and same number of cytosine (C) and guanine (G) bases. As a result, the
quantities of purine and pyrimidine bases will also be the same, with a 50% proportion for
each type. The rule A = T and C = G, or A/T = C/G = 1, is called Chargaff’s Rule, along with
the pairing rules described above.

In RNA there is only one nucleotide chain. RNA is a simple chain molecule and, as result,
there is no need for the proportions of the nitrogenous bases that compose it.

43) Which type of chemical bond maintains the pairing of each chain in the DNA molecule?
(C212.1) (PO1,2,3,4)
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To form the DNA molecule, purine bases bind to pyrimidine bases via intermolecular bonds
called hydrogen bonds. Hydrogen bonds occur when there is a hydrogen atom near one of the
following electronegative elements: fluorine, oxygen or nitrogen.

In such conditions, hydrogen appears to have lost electrons to those elements and a very
strong polarization is created. The highly positive hydrogen atom attracts pairs of electrons
from other molecules, making a hydrogen bond.

44) What is the name of the DNA duplication process? What is the main enzyme that is
involved in it? (C212.1) (PO1,2,3,4)

The process of copying, or duplication, of DNA molecules is called replication. The enzyme
involved in the formation of a new DNA chain is DNA polymerase. There are also other
important enzymes in the replication process, such as helicase, gyrase and ligase.

45) Why is the statement that DNA self-replicates incorrect? (C212.1) (PO1,2,3,4)

DNA is not completely autonomous in its replication process because the replication does not
occur without enzymatic activity. Therefore, it is not entirely correct to claim that DNA self-
replicates.

46) How do the two complementary nucleotide chains of DNA facilitate the replication
process of the molecule? (C212.1) (PO1,2,3,4)

The fact that the DNA molecule is made of two polynucleotide chains whose nitrogenous
bases form hydrogen bonds facilitates the replication of the molecule. During DNA
replication, the bond between the two chains is broken and each of them serves as a template
for the formation of a new nucleotide sequence along it, with the help of the enzyme DNA
polymerase and obeying the pairing rule A-T, C-G. At the end of the process, two double
helices of DNA are produced, each made of an original template chain and of a new
synthesized polynucleotide chain.

47) Which chemical bonds in DNA molecules must be broken for replication to occur?
(C212.1) (PO1,2,3,4)

During the DNA replication process, the hydrogen bonds between the nitrogenous bases of
the polynucleotide chains are broken.

48) As a result of DNA replication, two DNA molecules come into existence. G.
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is it
not correct to claim that two “new” DNA molecules are created? What is the name
given to this? (C212.1) (PO1,2,3,4)

During replication, each chain of the DNA molecule acts by pairing new nucleotides and,
after the process, two newly formed chains made from the bond between these nucleotides
appear. As a result, two DNA molecules are created, each with one chain from the original
molecule and one new chain formed by new nucleotides. Therefore, it is not entirely correct
to claim that the replication produces two new molecules of DNA. It is better to state that two
new half-molecules are created.

Because of this phenomenon, DNA replication is called semiconservative replication.

49) One characteristic of DNA molecules is their replication capability. What are the
consequences of failures during DNA replication? (C212.1) (PO1,2,3,4)

Ideally, a DNA molecule should replicate perfectly. However, sometimes failures in the
replication occur, causing the alteration (deletion, addition or substitution) of one or more
nucleotides in the molecule.

These mistakes, or mutations, also make changes in the protein synthesis process. For
example, the production of an important protein for cells or tissues may be suppressed; new
useful or unusable proteins may be created, etc. Mistakes in DNA replication and the
resulting creation of altered genetic material are some of the main creative forces behind
biological evolution and the diversity of species.

50) Where can RNA be found within cells? (C212.1) (PO1,2,3,4)

In the nucleus of eukaryotic cells, RNA can be found in the nuclear fluid along with DNA,
and is also the main component of the nucleolus. In the cytosol (in eukaryotes or in bacteria),
RNA molecules can be found on their own, as a structural component of ribosomes
(organelles specialized in protein synthesis) or even bound to them during the protein-making
process. Mitochondria and chloroplasts also have their own DNA and RNA.

UNIT II
DNA REPLICATION & REPAIR

1. What is the role of DNA ligase during replication. (C212.2) (PO1,2,3,4) (May 2019)
DNA ligase is an enzyme that repairs irregularities or breaks in the backbone of
double-stranded DNA molecules. It has important role in the process of DNA replication and
DNA repair. It has three general functions: It seals repairs in the DNA, it
seals recombination fragments, and it connects Okazaki fragments(small DNA fragments
formed during the replication of double-stranded DNA). DNA ligase functions G. by forming
Gomathia
bond between the end of a “donor” nucleotide and the end of an “acceptor” nucleotide.
 2. Explain the central dogma of molecular biology. (C212.2) (PO1,2,3,4)

The ‘Central Dogma’ is the process by which the instructions in DNA are converted into
a functional product. It was first proposed in 1958 by Francis Crick, discoverer of the structure of
DNA. The central dogma of molecular biology explains the flow of genetic information,
from DNA or RNA, to make a functional product, a protein. The central dogma suggests that
DNA contains the information needed to make all of our proteins, and that RNA is a messenger
that carries this information to the ribosomes. The ribosomes serve as factories in the cell where
the information is ‘translated’ from a code into the functional product. The process by which the
DNA instructions are converted into the functional product is called gene expression. Gene
expression has two key stages - transcription and translation. In transcription, the information in
the DNA of every cell is converted into small, portable RNA messages. During translation, these
messages travel from where the DNA is in the cell nucleus to the ribosomes where they are
‘read’ to make specific proteins. The central dogma states that the pattern of information that
occurs most frequently in our cells is:
From existing DNA to make new DNA (DNA replication)
From DNA to make new RNA (transcription)
From RNA to make new proteins (translation).

3. Differentiate prokaryotic and eukaryotic chromosome. (C212.2) (PO1,2,3,4)

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4. Define replication in DNA. (C212.2) (PO1,2,3,4)
DNA replication is the process by which a double-stranded DNA molecule is copied
to produce two identical DNA molecules. Replication is an essential process because,
whenever a cell divides, the two new daughter cells must contain the same genetic
information, or DNA, as the parent cell.
The replication process relies on the fact that each strand of DNA can serve as a template for
duplication. DNA replication initiates at specific points, called origins, where the DNA
double helix is unwound. A short segment of RNA, called a primer, is then synthesized and
acts as a starting point for new DNA synthesis. An enzyme called DNA polymerase next
begins replicating the DNA by matching bases to the original strand. Once synthesis is
complete, the RNA primers are replaced with DNA, and any gaps between newly synthesized
DNA segments are sealed together with enzymes.

3. Explain the bi directional mode of replication in DNA. (C212.2) (PO1,2,3,4)

DNA is double stranded molecule. Only one strand codes for proteins at any given
point in the molecule. However, both strands are used during DNA replication. Each of the
four bases in DNA (adenine, thymine, guanine, and cytosine) binds to a unique
complementary base on the other strand. Therefore the base sequence on one strand
determines the complementary sequence on the other strand. During DNA replication the two
strand separate from one another and each strand has a new complementary strand built onto
it. This form of replication is called bi directional; also called as semi conservative; each new
DNA molecule is composed of one conserved strand from the original molecule and one new
strand.

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4. What are called okazaki fragments? (C212.2) (PO1,2,3,4)
Okazaki fragments are the Short segments of DNA, 1000 to 2000 bases long, that
later join up to form continuouslengths of DNA. Okazaki fragments occur in replicating DNA
in both prokaryotes and eukaryotes. They form up on the‘lagging’ strand during replications
and join by ligation. (Reiji Okazaki, Japanese geneticist.) They are the DNA sequences, 100
to 200 nucleotides long, synthesized on the lagging strand of DNA in DNA replication.
Thefragments are subsequently ligated together to form a continuous strand. They are
produced because of the need forDNA polymerase to always synthesize in a 5′ to 3′ direction.

5. Explain the fidelity of DNA. (C212.2) (PO1,2,3,4)

The fidelity of a DNA polymerase is the result of accurate replication of a desired
template. Specifically, this involves multiple steps, including the ability to read a template
strand, select the appropriate nucleoside triphosphate and insert the correct nucleotide at the
3’ primer terminus, such that Watson-Crick base pairing is maintained. To effectively
discriminate correct vs. incorrect nucleotide incorporation, some DNA polymerases possess a
3’ to 5’ exonuclease activity. This activity, known as “proofreading,” is used to excise
incorrectly incorporated mononucleotides, which are then replaced with the correct
nucleotides. High- fidelity PCR uses DNA polymerases that couple low misincorporation
rates with proofreading to give faithful replication of the target DNA of interest.

6. When is fidelity important? (C212.2) (PO1,2,3,4)

When designing your PCR experiment, the first question you should ask is whether or
not your application requires a high-fidelity polymerase. If the outcome of your experiment
depends on the correct DNA sequence (e.g., cloning or next-generation sequencing
applications), you’ll want to minimize the incorporation of mismatched nucleotides by using
a high-fidelity polymerase. Fidelity is less important for standard PCR or colony PCR to
determine the presence or absence of an amplicon or to confirm that your plasmid has an
insert. Because of the robust nature of certain high-fidelity polymerases, some researchers use
them for all their amplifications, regardless of the PCR product’s downstream use.

7. How do you measure fidelity? (C212.2) (PO1,2,3,4)

Vendors use a variety of different methods to determine the fidelity of their DNA
polymerases. One assay, first described by Thomas Kunkel, uses portions of the lacZα gene
in M13 bacteriophage to correlate host bacterial colony color changes with errors in DNA
synthesis. Building on the Kunkel assay, Wayne Barnes’ assay is a common permutation
found in labs, in which PCR is used to copy the entire lacZ gene and portions of two drug-
resistance genes, with subsequent ligation, cloning, transformation and blue/white-colony
color determination. The readout of both assays is a white-colony phenotype caused by the
disruption of β-galactosidase activity that results from errors in the lacZ gene. With these
lacZ-based experimental approaches, the percentage of white colonies must be converted to
the number of errors per base incorporated. For a more direct readout of fidelity, Sanger
sequencing of individual cloned PCR products also can be used to score DNA polymerase
fidelity and offers the advantage of detecting all mutations. Using this method, the entire
mutational spectrum of a polymerase can be determined, and there is no need to correct for
nonphenotypic changes.

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8. List out certain inhibitors of DNA replication(C212.2) (PO1,2,3,4)
Alkylating antineoplastic agents, Nitrogen mustards, Topoisomerase inhibitors,
Altretamine, Bleomycin, Dacarbazine, Dactinomycin, Mitobronitol, Mitomycins, Mitosene,
Pingyangmycin, Plicamycin, Procarbazine, Temozolomide

9. Differntiate Eukaryotic and prokaryotic replication. (C212.2) (PO1,2,3,4)

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10. Define telomere. (C212.2) (PO1,2,3,4)
Telomeres are an essential part of human cells that affect how our cells age.
Telomeres are the caps at the end of each strand of DNA that protect our chromosomes, like
the plastic tips at the end of shoelaces. Without the coating, shoelaces become frayed until
they can no longer do their job, just as without telomeres, DNA strands become damaged and
our cells can’t do their job.

11. Describe the role of telomere in the replication. (C212.2) (PO1,2,3,4)

 DNA polymerase cannot replicate and repair DNA molecules at the ends of
linear chromosomes.
 The ends of linear chromosomes, called telomeres, protect genes from getting
deleted as cells continue to divide.
 The telomerase enzyme attaches to the end of the chromosome; complementary bases
to the RNA template are added on the 3' end of the DNA strand.
 Once the lagging strand is elongated by telomerase, DNA polymerase can add the
complementary nucleotides to the ends of the chromosomes and the telomeres can
finally be replicated.
 Cells that undergo cell division continue to have their telomeres shortened because
most somatic cells do not make telomerase; telomere shortening is associated with
aging.
 Telomerase reactivation in telomerase-deficient mice causes extension of
telomeres; this may have potential for treating age-related diseases in humans.

12. Describe the rolling circle mode of replication. (C212.2) (PO1,2,3,4)

Rolling circle replication is the unidirectional mode of DNA replication employed
by circular DNA molecules, such as plasmids and the genomes of bacteriophages and some
eukaryotic viruses. In viruses with linear genomes, the ability to circularise once inside a cell
is a
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crucial prerequisite for rolling circle replication. By replicating in this fashion, the virus can
ensure that no genetic material is lost from its genome as a consequence of successive rounds
of replication. Circularisation of linear phage genomes occurs by the interaction between cos
sites (cohesive sites) in the viral
genome.

The process begins with a plasmid or phage-encoded enzyme called relaxase, which creates a
nick in the circular DNA at a site called the double-strand origin (DSO); the relaxase remains
bound to the 5' phosphate at the site of this nick, so the 3' OH group is available as a primer
for DNA synthesis by DNA polymerase III. The polymerase moves along the nicked strand,
using the un-nicked strand as a template for replication, and a helicase displaces the nicked
strand behind polymerase as a single-stranded DNA molecule. This procedure can be
repeated multiple times to create numerous linear copies in a continuous head-to-tail series
called a concatemer.

To make these linear strands double-stranded and circular again, an initiator protein makes
another nick to terminate DNA synthesis. DNA polymerase III and RNA polymerase then
work in conjunction to replicate the single-strand origin (SSO) of a linear strand to make it
double- stranded. Finally, DNA polymerase I removes the primer, replacing it with DNA, and
DNA ligase covalently binds the strands end-to-end to make the final circular structure.

13. What is D- Loop replication? (C212.2) (PO1,2,3,4)

D-loop replication is a process by which chloroplasts and mitochondria replicate their
genetic material. An important component of understanding D-loop replication is that
manychloroplasts and mitochondria have a single circular chromosome like bacteria instead
of the linear chromosomes found in eukaryotes. However, many chloroplasts
andmitochondria have a linear chromosome, and D-loop replication is not important in these
organelles. In many organisms, one strand of DNA in the plastid comprises heavier
nucleotides (relatively more purines: adenine and guanine). This strand is called the H
(heavy) strand. The L (light) strand comprises lighter nucleotides (pyrimidines: thymine and
cytosine). Replication begins with replication of the heavy strand starting at the D-loop (also
known as the control region). An origin of replication opens, and the heavy strand is
replicated in one direction. After heavy strand replication has continued for some time, a new
light strand is also synthesized, through the opening of another origin of replication. When
diagramed, the resulting structure looks like the letter D. The D-loop region is important for
phylogeographic studies. Because the region does not code for any genes, it is free to vary
with only a few selective limitations on size and heavy/light strand factors. The mutation rate
is among the fastest of anywhere in either the nuclear or mitochondrial genomes in animals.
Mutations in the D-loop can effectively track recent and rapid evolutionary changes such as
within species and among very closely related species.

14. What are mutagens? (C212.2) (PO1,2,3,4)

Chemical mutagens are classified as alkylating agents, cross-linking agents,
and polycyclic aromatic hydrocarbons (PAHs). Alkylating agents act by adding molecular
components to DNA bases, which alters the protein product. Cross-linking agents create
covalent bonds with DNA bases, while PAHs are metabolized by the human body into other
potentially mutagenic molecules.
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Radiation is another potent mutagen. For biologists, the most significant forms of radiation
are light, heat, and ionizing radiation. Ionizing radiation can penetrate cells and create ions
in the cell contents. These, in turn, can cause permanent alterations in DNA; that is,
mutations. Ionizing radiation includes: x rays, gamma rays, and the subatomic particles—
neutrons, electrons ("beta" particles), and alpha particles (helium nuclei). Ionizing radiation
alters the way two strands of DNA interact. This high energy radiation passes through cells
and tissues, cutting up any DNA in its path. It can rearrange entire sections of the
chromosomes, altering relatively long stretches of DNA. UV radiation causes covalent bonds
to form between neighboring thymine bases in the DNA, so altering the DNA product at that
location.

15. Explain the importance of DNA repair mechanism. (C212.2) (PO1,2,3,4)

DNA in the living cell is subject to many chemical alterations (a fact often forgotten
in the excitement of being able to do DNA sequencing on dried and/or frozen specimens. If
the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes
must be corrected. A failure to repair DNA produces a mutation. The recent publication
of the human genome has already revealed 130 genes whose products participate in DNA
repair. More will probably be identified soon.

16. What are the agents that can damage DNA? (C212.2) (PO1,2,3,4)
 Agents that Damage DNA
 Certain wavelengths of radiation
 ionizing radiation such as gamma rays and X-rays
 Ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by
DNA but also the longer-wavelength UV-B that penetrates the ozone shield.
 Highly-reactive oxygen radicals produced during normal cellular respiration as well
as by other biochemical pathways.
 Chemicals in the environment
 many hydrocarbons, including some found in cigarette smoke
 some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
 Chemicals used in chemotherapy, especially chemotherapy of cancers

17. What are the Types of DNA Damage? (C212.2) (PO1,2,3,4)

All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.
One of the most frequent is the loss of an amino group ("deamination") — resulting, for
example, in a C being converted to a U. Mismatches of the normal bases because of a failure
of proofreading during DNA replication. Common example: incorporation
of the pyrimidine U (normally found only in RNA) instead of T. Breaks in the backbone. Can
be limited to one of the two strands (a single-stranded break, SSB) or on both strands (a
double- stranded break (DSB). Ionizing radiation is a frequent cause, but some chemicals
produce breaks as well. Crosslinks Covalent linkages can be formed between bases on the
same DNA strand ("intrastrand") or on the opposite strand ("interstrand").

18. Explain the Meselson–Stahl experiment(C212.2) (PO1,2,3,4)

The Meselson-Stahl was an experiment by Matthew Meselson and Franklin Stahl
with some additional help from a Canadian biologist, Mason MacDonald, and Indian-
Canadian nuclear physicist, Amandeep Sehmbi, in 1958 which supported the hypothesis that
DNA
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 replication was semiconservative. In semiconservative replication, when the double stranded
DNA helix is replicated, each of the two new double-stranded DNA helices consisted of one strand from
the original helix and one newly synthesized. It has been called "the most beautiful experiment in
biology. Meselson and Stahl decided the best way to tag the parent DNA would be to change one of the
atoms in the parent DNA molecule. Since nitrogen is found in the nitrogenous bases of each nucleotide,
they decided to use an isotope of nitrogen to distinguish between parent and newly copied DNA. The
isotope of nitrogen had an extra neutron in the nucleus, which made it heavier.

19. What are the enzymes that contribute for excision repair mechanisms? (C212.2)
(PO1,2,3,4)
 DNA glycosylases.
 AP endonucleases.
 End processing enzymes.
 DNA polymerases.
 Flap endonuclease.
 DNA ligase
 MBD4.
 NEIL1.

20. List the natural agents that commonly cause damage in our DNA. (C212.2) (PO1,2,3,4)
Water (deamination, depurination), oxygen (through the superoxide radical, hydrogen
peroxide, and hydroxyl radical).

21. Outline the principles involved in eukaryotic DNA synthesis. (C212.2) (PO1,2,3,4)
Restriction to S phase, semi-conservative replication, initiation at internal origins,
simultaneous replication of many chromosomal segments as they move through
polymerization sites in factories, strand separation to give a replication bubble flanked by two
replication forks, requirements for primers and a primase, growth 5'-to-3', continuous and
discontinuous synthesis on leading and lagging strands.

22. How would you demonstrate that active DNA polymerases are fixed to an
underlying structure in the nucleus? (C212.2) (PO1,2,3,4)
Permeabilize cells, treat -/+ nuclease, remove detached chromatin, measure remaining
polymerizing activity by incorporation of radiolabeled dTTP; removing most chromatin
leaves most activity.

23. Outline the different approaches used to label sites of DNA synthesis in eukaryotic
nuclei, and the difficulties associated with each one. (C212.2) (PO1,2,3,4)
By immunolabeling polymerases: not all enzyme active. By autoradiography with
[3H]thymidine: slow entry and conversion to immediate precursor, dilution by endogenous
pools, complications of rapidity of DNA synthesis, long path-length of -particles. By
immunolabeling after incubation with Br-dU: slow entry and conversion, but higher
resolution afforded by immuno-EM. By immunolabelling after permeabilization with
immediate precursors like Br- dUTP and biotin-dUTP: control of elongation rate, but lysis
might alter structure, and still limited resolution (even with immunogold labeling). Details of
factories best seen after removing most chromatin.
24. What is the unwinding problem, and how might it be solved in theory and in
practice? (C212.2) (PO1,2,3,4) G. Gomathi
Each strand in a DNA duplex is entwined about its partner, and must be
untwined during replication. Theoretical solutions: by rotation about ends (but it these are
fixed the two strands remain interlocked), by cutting one or other of the strands (or both),
passing one (or both) strands through the break, and resealing the break. Practical
solution: topoisomerases cut, pass, and reseal.

25. Describe the structure of the origin of replication in E. coli. (C212.2) (PO1,2,3,4)
OriC contains: four 9-mers containing a specific recognition sequence (i.e., 5'-
TTAT(C/A)CA(C/A)) for the initiator protein dnaA, three 13-mers that melt easily, 11
potential sites (i.e., GATC) of methylation by the Dam methylase, and 2 back-to-back
promoters that may be involved in the initiation of replication.

26. What is an 'autonomously-replicating sequence' (ARS)? How was the first one
identified in yeast? (C212.2) (PO1,2,3,4)
ARS: DNA sequence that enables circular plasmid lacking origin to replicate in
yeast cells, usually equivalent to an origin of replication. Discovery: The first ARS was
obtained as follows. Yeast mutants lacking the LEU gene cannot form colonies without
added leucine. Even on transformation with a bacterial plasmid carrying the yeast LEU+
gene, few colonies result; this is because the plasmid is unable to replicate along with the
yeast chromosomes and is soon diluted out. However, if random pieces of yeast DNA are
inserted into the plasmid, a few will now contain a yeast replication origin and so can
replicate in yeast cells. Cells carrying such a plasmid will grow into a colony since they
contain both the LEU+ gene and a yeast origin that facilitates plasmid replication.

27. Outline the problem associated with replicating the ends of a chromosome, and
some solutions. (C212.2) (PO1,2,3,4)
A polymerase can extend a leading strand to the very end, but removal of a
primer at the 5' end of the lagging strand leaves a gap that cannot be filled, as no 3'OH is
available. Solutions: use protein-nucleotide priming (adenovirus), form a hairpin,
concatamer, or circle (e.g., in vaccinia, T7 and lambdoid viruses), maintain ends by
recombination (e.g., T4 bacteriophage), use telomerase.

28. Outline the properties of telomerase. (C212.2) (PO1,2,3,4)

It is part protein and part RNA, protein part has homology with reverse
transcriptases, RNA part contains 8-30 nucleotides of RNA containing 1.2-1.9 copies of
the C-strand repeat that templates synthesis of telomeric DNA.

29. How were replication factories imaged in B. subtilis? (C212.2) (PO1,2,3,4)

Using a PolC-GFP construct - one discrete spot is generally seen in the middle of the cell.

30. What is a proofreading activity? (C212.2) (PO1,2,3,4)

A 3'->5' exonuclease (either part of the catalytic subunit of a DNA polymerase,
or a subunit of the polymerizing complex) that removes mispaired bases immediately
after they have been incorporated.

32) Define leading & lagging strand. (C212.2) (PO1,2,3,4)

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32) Explain role of DNA helicase. (C212.2) (PO1,2,3,4)
Helicases are enzymes that bind and may even remodel nucleic acid or nucleic acid protein
complexes. There are DNA and RNA helicases. DNA helicases are essential during DNA replication
because they separate double-stranded DNA into single strands allowing each strand to be copied.
During DNA replication, DNA helicases unwind DNA at positions called origins where synthesis
will be initiated. DNA helicase continues to unwind the DNA forming a structure called the
replication fork, which is named for the forked appearance of the two strands of DNA as they are
unzipped apart. The process of breaking the hydrogen bonds between the nucleotide base pairs in
double-stranded DNA requires energy. To break the bonds, helicases use the energy stored in a
molecule called ATP, which serves as the energy currency of cells. DNA helicases also function in
other cellular processes where double-stranded DNA must be separated, including DNA repair and
transcription. RNA helicases are involved in shaping the form of RNA molecules, during all
processes involving RNA, such as transcription, splicing, and translation.

34) Explain termination event in DNA replication. (C212.2) (PO1,2,3,4)

DNA replication can be divided into three distinct steps: initiation, elongation, and
termination. The bidirectional replication of a circular chromosome of bacteria terminates at a
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position where the two replication forks meet. Bacteria have developed a system that ensures
termination will occur within a restricted terminus region. This is achieved by a combination of a
 DNA motif of 20 to 30 bp, called the ter sequence, and a cognate termination protein that recognizes
ter sites and binds to them tightly.

35) Define substitution mutation with sub types. (C212.2) (PO1,2,3,4)

A substitution is a mutation that exchanges one base for another (i.e., a change in a single "chemical letter" such as
switching an A to a G). Such a substitution could:
1. change a codon to one that encodes a different amino acid and cause a small change in the protein produced.
For example, sickle cell anemia is caused by a substitution in the beta-hemoglobin gene, which alters a
single amino acid in the protein produced.
2. change a codon to one that encodes the same amino acid and causes no change in the protein produced.
These are called silent mutations.

3. change an amino-acid-coding codon to a single "stop" codon and cause an incomplete protein. This can
have serious effects since the incomplete protein probably won't function.

36) Draw a diagram of Okazaki fragment formation. (C212.2) (PO1,2,3,4)

37) Comment on role of dnaA & dnaB protein in DNA replication. (C212.2) (PO1,2,3,4)
DnaA is a protein that activates initiation of DNA replication in bacteria.[1] It is a replication initiation
factor which promotes the unwinding of DNA at oriC.[1] The onset of the initiation phase of DNA
replication is determined by the concentration of DnaA. [1] DnaA accumulates during growth and then
triggers the initiation of replication. [1] Replication begins with active DnaA binding to 9-mer (9-bp)
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repeats upstream of oriC.[1] Binding of DnaA leads to strand separation at the 13-mer repeats. [1] This
binding causes the DNA to loop in preparation for melting open by the helicase DnaB.[1]
DnaB helicase is an enzyme in bacteria which opens the replication fork during DNA replication. Although the
mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is
unknown, a change in the quaternary structure of the protein involving dimerisation of the N-terminal domain has
been observed and may occur during the enzymatic cycle.[1]Initially when DnaB binds to dnaA, it is associated
with dnaC, a negative regulator. After DnaC dissociates, DnaB binds dnaG.

38) Explain briefly process of Alkylation. (C212.2) (PO1,2,3,4)

Alkylation is the process of transferring an alkyl group from one molecule to another. Alkyl substituent is an alkane
which have one missing hydrogen atom. It is basically the process of introducing hydrocarbons into chemicals. An
alkyl group molecule has general formula as C nH2n+1, where ‘n’ signifies number of carbons associated together. An
alkyl group can be transferred as an alkyl free radical, carbonation, a carbanion or as a Carbene.
Alkyl groups can also be removed from the compound by the process Dealkylation.

39) Comment on direct repair mechanism. (C212.2) (PO1,2,3,4)

Direct reversal of DNA damage is a mechanism of repair that does not require a template and is applied to two
main types of damage. UV light induces the formation of pyrimidine dimers which can distort the DNA chain
structure, blocking transcription beyond the area of damage.

Direct reversal through photoreactivation can inverse this dimerization reaction by utilizing light energy for the
destruction of the abnormal covalent bond between adjacent pyrimidine bases. This type of photoreactivation does
not occur in humans.

The damage caused by alkylating agents reacting with DNA can also be repaired through direct reversal.
Methylation of guanine bases produces a change in the structure of DNA by forming a product that is
complimentary to thymine rather than cytosine. The protein methyl guanine methyl transferase (MGMT) can
restore the original guanine by transferring the methylation product to its active site.

40) Give role of DNA glycolysis & AP endonuclease in base excision repair. (C212.2) (PO1,2,3,4)

a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of
DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed.
It is proposed that related systems are ubiquitous.
Apurinic/apyrimidinic (AP) endonuclease is an enzyme that is involved in the DNA base excision repair pathway
(BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in
the phosphodiester backbone of the AP site created when DNA glycosylase removes the damaged base.

41) What is oxidative deamination? Explain it. (C212.2) (PO1,2,3,4)

Oxidative deamination is a form of deamination that generates α-keto acids and other oxidized products from
amine-containing compounds, and occurs largely in the liver and kidney. Oxidative deamination is an important
step in the catabolism of amino acids, generating a more metabolizable form of the amino acid, and also
generating ammonia as a toxic byproduct. The ammonia generated in this process can then be neutralized
into urea via the urea cycle.
G. Gomathi
42) Explain activities of DNA polymerase – I(C212.2) (PO1,2,3,4)
DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication.
Discovered by Arthur Kornberg in 1956,[1] it was the first known DNA polymerase (and the first known of any kind
of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other
bacteria, the gene that encodes Pol I is known as polA. The E. coliform of the enzyme is composed of 928 amino
acids, and is an example of a processive enzyme—it can sequentially catalyze multiple polymerisations without
releasing the single-stranded template.[2] The physiological function of Pol I is mainly to repair any damage with
DNA, but it also serves to connect Okazaki fragments by deleting RNA primers and replacing the strand with
DNA.

43) Describe Okazaki fragment formation(C212.2) (PO1,2,3,4)

During DNA replication, the double helix is unwound and the complementary strands are separated by the
enzyme DNA helicase, creating what is known as the DNA replication fork. Following this fork, DNA primase and
then DNA polymerase begin to act in order to create a new complementary strand. Because these enzymes can only
work in the 5’ to 3’ direction, the two unwound template strands are replicated in different ways. [2] One strand,
the leading strand, undergoes a continuous replication process since its template strand has 3’ to 5’ directionality,
allowing the polymerase assembling the leading strand to follow the replication fork without interruption. The
lagging strand, however, cannot be created in a continuous fashion because its template strand has 5’ to 3’
directionality, which means the polymerase must work backwards from the replication fork. This causes periodic
breaks in the process of creating the lagging strand. The primase and polymerase move in the opposite direction of
the fork, so the enzymes must repeatedly stop and start again while the DNA helicase breaks the strands apart.
Once the fragments are made, DNA ligase connects them into a single, continuous strand. [3] The entire replication
process is considered "semi-discontinuous" since one of the new strands is formed continuously and the other is
not.[4.

44) Write short note on Base analogue (C212.2) (PO1,2,3,4)

Chemicals that resemble nucleotide bases are called base analogues which can be incorporated into a growing
nucleoide chain. They often generate mutations of the polyadenylation site. E.g. bromodeoxyuracil.

45) Explain ‘Dark repair mechanism’(C212.2) (PO1,2,3,4)

Mutation or damage to DNA occurs in several ways. To survive the damaging effect of the mutation, cells possess
mechanisms to repair the DNA. Repair may involve either the reversal of the process that led to mutation or the
actual removal of the damaged part.
A process known as “dark repair or excision repair process” removes the damaged part and restores the
original structure. In this process the nicks or hydrolyzes the Phospodiester bond at the 5’ end of the thymidine
dimer. Then an exonuclease removes the dimer from the strand. The gap that is formed is filled up by reinsertion of
appropriate nucleotide. DNA polymerase I and DNA ligase complete the above reactions.

46) Compare: Natural & Artificial mutation. (C212.2) (PO1,2,3,4)

BASIS FOR NATURAL ARTIFICIAL
COMPARISON SELECTION SELECTION

Meaning Natural selection Artificial selection

involves the natural involves the artificial
process of selection, process where selection
favouring the most is done by favouring the
fittest which is able to desired characters in the
face all types of new organisms. G. Gomathi
situations.
 BASIS FOR NATURAL ARTIFICIAL
COMPARISON SELECTION SELECTION

Chances of Chances of surviving Selection of characters is

survival increased. done artificially hence
chances of surviving of
new breed is at risk
whether it is a plant, or
animals or any other
organism.

Process rate Slow and long process. Faster.

Controlled by Controlled by nature. Artificial selection is

controlled by humans.

Performed on Natural selection is Artificial selection is

performed on all types processed on some
of organisms. selective organisms of
humans desires.

Selection of Selection is totally Selection is done on the

traits based on the adaptable basis of required
character and the one character.
who is able to cop up in
all types of natural
conditions.

Transformation It transforms the entire It brings out the new

population of a species. variety of that species.

Type of Natural selection. Man-made selection.

selection

Occurs in It occurs in all kind of It usually occurs in

natural populations. domestic populations.

Examples Darwin finches which It is usually performed on

are group of birds of 14 pet animals or animals
species of small birds, which are used for
evolved from the same economical purposes.
species of bird on the
Galapagos Islands.

G. Gomathi
47) Write a note on structure of DNA polymerase – III(C212.2) (PO1,2,3,4)

48) Explain the initiation event in prokaryotic DNA replication. (C212.2) (PO1,2,3,4)
All cells must finish DNA replication before they can proceed for cell division. Media conditions that support fast
growth in bacteria also couples with shorter inter-initiation time in them, i.e. the doubling time in fast growing cells
is less as compared to the slow growth. [5] In other words, it is possible that in fast growth conditions the
grandmother cells starts replicating its DNA for grand daughter cell. For the same reason, G. the Gomathi
initiation of DNA
replication is highly regulated. Bacterial origins regulate orisome assembly, a nuclei-protein complex assembled on
the origin responsible for unwinding the origin and loading all the replication machinery. In E. coli, the direction
for orisome assembly are built into a short stretch of nucleotide sequence called as origin of replication (oriC)
which contains multiple binding sites for the initiator protein DnaA [6] (a highly homologous protein amongst
bacterial kingdom). DnaA has four domains with each domain responsible for a specific task. [7] There are 11 DnaA
binding sites/boxes on the E. coli origin of replication [6] out of which three boxes R1, R2 and R4 (which have a
highly conserved 9 bp consensus sequence 5' - TTATC/ACACA [2]) are high affinity DnaA boxes. They bind to
DnaA-ADP and DnaA-ATP with equal affinities and are bound by DnaA throughout most of the cell cycle and
forms a scaffold on which rest of the orisome assembles. The rest eight DnaA boxes are low affinity sites that
preferentially bind to DnaA-ATP.[6] During initiation, DnaA bound to high affinity DnaA box R4 donates additional
DnaA to the adjacent low affinity site and progressively fill all the low affinity DnaA boxes. [6] Filling of the sites
changes origin conformation from its native state. It is hypothesized that DNA stretching by DnaA bound to the
origin promotes strand separation which allows more DnaA to bind to the unwound region. [8] The DnaC helicase
loader then interacts with the DnaA bound to the single-stranded DNA to recruit the DnaB helicase,[9] which will
continue to unwind the DNA as the DnaG primase lays down an RNA primer and DNA Polymerase III
holoenzyme begins elongation.

49) Define Chemical & Physical mutagen. (C212.2) (PO1,2,3,4)

50) Explain role of single strand binding protein in DNA replication. (C212.2) (PO1,2,3,4)

Single-strand DNA-binding protein (SSB) is a protein found in Escherichia coli (E. coli) bacteria, that binds to
single-stranded regions of deoxyribonucleic acid (DNA).[1] Single-stranded DNA is produced during all aspects of
DNA metabolism: replication, recombination, and repair. As well as stabilizing this single-stranded DNA, SSB
proteins bind to and modulate the function of numerous proteins involved in all of these processes.

UNIT III – TRANSCRIPTION

1. What are the basic characteristic of prokaryotic promoter (May2019)
Prokaryotic promoters
In prokaryotes, the promoter consists of two short sequences at -10 and G. -35 Gomathi
positions
upstream from the transcription start site. The sequence at -10 is called the Pribnow box,
or the - 10 element, and usually consists of the six nucleotides TATAAT. The Pribnow
 box is absolutely essential to start transcription in prokaryotes. The other sequence at -35
(the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a
very high transcription rate
2. What is the significance of 5’-cappingof mRNA?(May 2019)
a. Capping prevents 5’ degradation from 5’exonucleases.
b. Capping provides stability to mRNAs.
c. Capping facilitates the transport of mRNA into cytoplasm otherwise they remain
in the nucleus.
d. Capping enhances the efficiency of translation of mRNAs.
e. Capping enhances the efficiency of splicing at 5’end introns.
f. Capping with poly (A) provides synergism during translation.

3. Define bacterial transformation. (C212.3) (PO1,2,3,4)

Bacterial transformation the exchange of genetic material between strains of bacteria
by the transfer of a fragment of naked DNA from a donor cell to a recipient cell, followed by
recombination in the recipient chromosome.

4. What are the properties of genetic material? (C212.3) (PO1,2,3,4)

a. Stores genetic information
b. Physical and Chemical stability (The double stranded protects the DNA from
chemical attack)
c. Able to undergo mutation
d. Stored information accessible to progeny

5. Describe the basic rule for the replication of all nucleic acids. (C212.3) (PO1,2,3,4)
a. The primary role of any mode of replication is to duplicate the base sequence of
the parent molecule. The specificity of base pairing adenine with thymine and
guanine with cytosine provides the mechanism used by all replication systems.
b. Nucleotide monomers are added one by one to the end of a growing strand by an
enzyme called a DNA polymerase.
c. The sequence of bases in each new or daughter strand is complementary to the
base sequence in the original template or parent strand being copied that is, if
there is an adenine in the parent strand, a thymine nucleotide will be added to the
end of the growing daughter strand when the adenine is being copied.

6. Define a TATA box. (C212.3) (PO1,2,3,4)

The TATA box (also called Goldberg-Hogness box) is a DNA sequence (cis-
regulatory element) found in the promoter region of genes in archaea and eukaryotes;
approximately 24% of human genes contain a TATA box within the core promoter

7. Explain any four general features of enhancers(C212.3) (PO1,2,3,4)

Several DNA sequences of note have been detected in eukaryotic genes. The first that
was described was an enhancer sequence. Enhancers have the ability to greatly increase the
expression of genes in their vicinity

8. Define genetic code. (C212.3) (PO1,2,3,4)

The genetic code is the set of rules by which information encoded in genetic material
(DNA or mRNA sequences) is translated into proteins (amino acid sequences) byG. living cells.
Gomathi
9. Give any two inhibitors of protein synthesis in eukaryotes along with its action(C212.3)
(PO1,2,3,4)
 Protein synthesis is a complex, multi-step process involving many enzymes as well as
conformational alignment. However, the majority of antibiotics that block bacterial protein
synthesis interfere with the processes at the 30S subunit or 50S subunit of the 70S bacterial

G. Gomathi
ribosome. The aminoacyltRNA synthetases that activate each amino acid required for peptide
synthesis are not antibiotic targets. Instead, the primary steps in the process that are attacked
are
a. The formation of the 30S initiation complex (made up of mRNA, the 30S
ribosomal subunit, and formyl-methionyl-transfer RNA),
b. The formation of the 70S ribosome by the 30S initiation complex and the 50S
ribosome, and
c. The elongation process of assembling amino acids into a polypeptide.

10. Why genetic activity is regulated? (C212.3) (PO1,2,3,4)

a. To discuss the structure and transcription of bacterial gene
b. To describe the molecular mechanism and to regulate gene activity

11. What is temperature sensitive mutation? (C212.3) (PO1,2,3,4)

A viral mutant that is able to replicate at one portion of a temperature range but not at
another, the parent (wild type) strain being able to replicate over the whole temperature
range.

12. Explain cantenation. (C212.3) (PO1,2,3,4)

Catenation is the ability of a chemical element to form a long chain-like structure via
a series of covalent bonds. Catenation occurs most readily in carbon, which forms covalent
bonds with other carbon atoms

13. What are Satellite DNA? (C212.3) (PO1,2,3,4)

Satellite DNA consists of very large arrays of tandemly repeating, non-coding DNA.
Satellite DNA is the main component of functional centromeres, and form the main structural
constituent of heterochromatin. The name "satellite DNA" refers to how repetitions of a short
DNA sequence tend to produce a different frequency of the nucleotides adenine, cytosine,
guanine and thymine, and thus have a different density from bulk DNA - such that they form
a second or 'satellite' band when genomic DNA is separated on a density gradient.

14. Define linkage(C212.3) (PO1,2,3,4)

Two genes are said to be under linkage, or linked, when they reside in the same
chromosome. For example, the research of the human genome discovered that the factor III
of clotting gene and the factor V of clotting gene are located in the same chromosome (the
human chromosome 1). The factor VII gene however is not linked to those genes since it is
located in the chromosome 13

15. Explain suppressor sensitive mutation. (C212.3) (PO1,2,3,4)

A conditionally lethal, host range, bacteriophage mutant that produces
nonsense codons and can replicate only in a host bacterium able to translate the nonsense
codon; the mutation's effects are lethal (i.e., prevent replication of the virus) in a bacterium
without such a suppressor mechanism.

16. What is breathing means in DNA structure? (C212.3) (PO1,2,3,4)

Recent claim is discussed that Watson-Crick pairs in the naked duplex DNA
spontaneously flip into Hoogsteen pairs under ordinary conditions. The claim is considered
within the historical retrospective and is put into the broader context of DNA biophysics.
G. Gomathi
17. Explain double sieve mechanism(C212.3) (PO1,2,3,4)
A model that explains the rarity of misacylation of amino acids by proposing that an
amino acid larger than the correct one is rarely activated because (1) it is too large to fit into
the active site of the tRNA synthetase (first sieving), and (2) the hydrolytic site of the same
synthetase is too small for the correct amino acid (second sieving). Thus, an amino acid
smaller than the correct one can be removed by hydrolysis.

18. Define cot value. (C212.3) (PO1,2,3,4)

Renaturation is a bimolecular reaction where the reaction rate is directly proportional
to the product of the concentrations of c of the two homologous DNA strands.
The renaturation rate is = dc/dt = K2[W]
[C] Integration of the above equation
gives;
C/Co = 1/(1+k2 C0t)
Where C is the concentration of single-stranded DNA at time t (in min), and C0 is the
concentration of DNA at time zero.C/C0 = ½, then K2 = 1/ C0t1/2

In other words, the product of the initial concentration of ssDNA, C0 and the time required to
renature 50% of the DNA, t1/2 is inversely proportional to the rate constant K2 of the
reaction. This C0t1/2 is called the Cot value. The Cot value is directly proportional to the
complexity of the genome.

19. Explain specialized transduction(C212.3) (PO1,2,3,4)

Specialized transduction - only specific regions of chromosome located near
attachment site are transduced, transducing particles carry both chromosomal DNA and
phage DNA.

20. What are simple multigene families? Give example. (C212.3) (PO1,2,3,4)
The term multigene families is used to include groups of genes from the same
organism that encode proteins with similar sequences either over their full lengths or limited
to a specific domain. DNA duplications can generate gene pairs. If both copies are
maintained in subsequent generations then a multigene family will exist. A multigene family
is a member of a family of related proteins encoded by a set of similar genes. Multigene
families are believed to have arisen by duplication and variation of a single ancestral gene.
Examples of multigene families include those that encode the actins, hemoglobins,
immunoglobulins, tubulins, interferons, histones etc.

21. Differentiate prokaryotic and eukaryotic promoters(C212.3) (PO1,2,3,4)

Prokaryotic promoters
In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions
upstream from the transcription start site. The sequence at -10 is called the Pribnow box, or
the - 10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is
absolutely essential to start transcription in prokaryotes. The other sequence at -35 (the -35
element) usually consists of the six nucleotides TTGACA. Its presence allows a very high
transcription rate.
Eukaryotic promoters
G. Gomathi
Eukaryotic promoters are extremely diverse and are difficult to characterize. They
typically lie upstream of the gene and can have regulatory elements several kilobases away
from
the transcriptional start site. In eukaryotes, the transcriptional complex can cause the DNA to
bend back on itself, which allows for placement of regulatory sequences far from the actual
site of transcription. Many eukaryotic promoters, contain a TATA box (sequence TATAAA),
which in turn binds a TATA binding protein which assists in the formation of the RNA
polymerase transcriptional complex. The TATA box typically lies very close to the
transcriptional start site (often within 50 bases).

22. What are the three enzymatic activities for DNA polymerase I? (C212.3) (PO1,2,3,4)
DNA Polymerase I (or Pol I) is an enzyme that participates in the process of
DNA replication and is exclusively found in prokaryotes. It is composed of 928 amino acids,
and is an example of a processive enzyme - it can sequentially catalyze multiple
polymerisations. Discovered by Arthur Kornberg in 1956,[1] it was the first known DNA
polymerase (and, indeed, the first known of any kind of polymerase). It was initially
characterized in E. coli, although it is ubiquitous in prokaryotes. In E. coli and many other
bacteria, the gene that encodes Pol I is known as polA.

23. Mention the beneficial effects of capping and tailing of RNA(C212.3) (PO1,2,3,4)
a. Capping prevents 5’ degradation from 5’exonucleases.
b. Capping provides stability to mRNAs.
c. Capping facilitates the transport of mRNA into cytoplasm otherwise they remain
in the nucleus.
d. Capping enhances the efficiency of translation of mRNAs.
e. Capping enhances the efficiency of splicing at 5’end introns.
f. Capping with poly (A) provides synergism during translation.
g. Luciferase mRNAs have been used to determine its half-life and translation
efficiency with or without cap and poly- (A) tail.
h. Half-life of Luciferase mRNA without cap and without poly (A) is just 31
minutes, and translational activity is 2900 (as measured in terms of light emitted
by ug of radioactive protein).
i. But mRNAs without cap but with poly (A) tail shows half-life of 44 minutes. And
its activity is 4480. The capped mRNA without poly (A) has half-life of 53
minutes and translation activity is 62000 a virtual 50% increase in its half-life and
translational efficiency.
j. The capped mRNA with poly- (A) tail, has a half-life of 100 minutes and its
translational activity is 1,333 000; the relative effect of cap on its activity 200
fold.
k. During translation mRNA cap and poly-A tail bind to each other through a protein
eF4G and gets circularized.

24. Outline the basic principles involved in eukaryotic RNA synthesis. (C212.3) (PO1,2,3,4)
Transcription between promoter (start) and termination (stop) signals, multi-subunit
polymerases in factories, the basic steps of transcription, initiation of synthesis of new chains,
synthesis 5'-to-3'.

25. How would you determine which parts of the genome are transcribed? (C212.3) (PO1,2,3,4)
'Miller' spreads, 'S1 mapping', and RT-PCR.

G. Gomathi
 26. Describe the properties of the bacterial RNA polymerase. (C212.3) (PO1,2,3,4)
The core enzyme (initiates poorly), σ (helps the core initiate), the holoenzyme, TATA
and -35 boxes, closed and open complexes, rho independent and dependent terminators.

27. What are the untwining and supercoiling problems, and how are they resolved? (C212.3)
(PO1,2,3,4)
Untwining problem (and solution): a tracking polymerase is likely to generate a
transcript that is entangled about the template (fix the polymerase and allow DNA to rotate).
Supercoiling problem (and solution): transcription by both tracking and fixed polymerases
generates twin domains of supercoiling (role of topoisomerase).

28. How is a 'Miller' spread prepared, and illustrate the appearance of a spread
containing some ribosomal cistrons? (C212.3) (PO1,2,3,4)
Preparation: isolate nuclei, disperse chromatin in a hypotonic solution, spin onto a grid.
Structure: series of 'Christmas' trees.

29. How can caps be isolated? (C212.3) (PO1,2,3,4)

Exhaustively treat mRNA with endonucleases that cleave 3' phosphates next to bases
(e.g., RNase T2) to leave 5' -> 5' links intact; purify resulting dinucleotides (each carrying
several phosphates) free of mononucleotides on a column (separate molecules carrying
different numbers of phosphate groups).

30. What is the role of the cap? (C212.3) (PO1,2,3,4)

The cap binds the cap-binding complex, CBC, which tethers the nascent transcript to
the factory, enhances 3' end formation, protects transcripts from degradation, facilitates
export from the nucleus, and dissociates at the ribosome to be replaced by the translational
regulator, eIF-4E.

31. What is 'nonsense mediated decay' (NMD), and how was it discovered? (C212.3) (PO1,2,3,4)
NMD: mRNA with a stop codon in the normal position is stable in both nucleus and
cytoplasm, but moving the stop codon near the 5' end leads to the loss - or NMD - of the
message. Discovery: place stop codons at different positions in test genes (e.g., TPI) and then
monitor transcript levels; stop codons close to the 5' end destabilize the transcript in both the
nucleus and cytoplasm.

32. What is 'transcriptional interference', and how was it discovered? (C212.3) (PO1,2,3,4)
Transcriptional interference: phenomenon where transcription of one gene prevents
transcription of an adjacent gene. Discovery: Cells were transfected with a retroviral vector
encoding resistance to neomycin and azaguanine, and clones harboring a single copy of the
vector selected. Expression of the 3' gene was suppressed when selection required expression
of the 5' gene, and vice versa. In addition, hardly any cells grew in both neomycin and
azaguanine.

31) What is the role of sigma factor in transcription. (C212.3) (PO1,2,3,4)

A sigma factor (σ factor) is a protein needed for initiation of transcription in bacteria.[1] It is a bacterial
transcription initiation factor that enables specific binding of RNA polymerase(RNAP) to gene promoters. It is
homologous to archaeal transcription factor B and to eukaryotic factor TFIIB.[2] The specificG.sigma
Gomathi
factor used to
initiate transcription of a given gene will vary, depending on the gene and on the environmental signals needed to
initiate transcription of that gene. Selection of promoters by RNA polymerase is dependent on the sigma factor that
associates with it.
32)

33) What is the role of RNA polymerase? (C212.3) (PO1,2,3,4)

RNAP locally opens the double-stranded DNA (usually about four turns of the double helix) so that one strand of
the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription

34) Draw the structure of mRNA and explain it. (C212.3) (PO1,2,3,4)
Messenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from DNA to
the ribosome, where they specify the amino acid sequence of the protein products of gene expression. RNA
polymerase transcribes primary transcript mRNA (known as pre-mRNA) into processed, mature mRNA. This
mature mRNA is then translated into a polymer of amino acids: a protein, as summarized in the central dogma of
molecular biology.

35) Explain 80s ribosome of eukaryotes. (C212.3) (PO1,2,3,4)

Eukaryotic ribosomes are also known as 80S ribosomes, referring to their sedimentation coefficients in Svedberg
units, because they sediment faster than the prokaryotic (70S) ribosomes. Eukaryotic ribosomes have two unequal
subunits, designated small subunit (40S) and large subunit (60S) according to their sedimentation coefficients. Both
subunits contain dozens of ribosomal proteins arranged on a scaffold composed of ribosomal RNA (rRNA). The
small subunit monitors the complementarity between tRNA anticodon and mRNA, while the large subunit
catalyzes peptide bond formation.

36) Write a note on initiation of translation. (C212.3) (PO1,2,3,4)

The initiation of protein synthesis consists in the recruitment of a ribosome·initiator tRNA complex to the initiation
codon of a messenger RNA. In prokaryotes, this process involves the direct interaction of the ribosomal RNA with
the mRNA. In contrast, eukaryotes have evolved a sophisticated mechanism that relies mostly on protein-RNA and
protein-protein interactions. Eukaryotes have taken advantage of the evolution of novel mRNA structures, such as
the 5' cap and the poly (A) tail, to develop new mechanisms for the recruitment of the ribosome to the mRNA. As a
result, the eukaryotic translation initiation apparatus is now a complex machinery comprising at least eleven
factors. This complexity provides a fertile ground for enhanced regulation, and many new mechanisms have been
adopted by eukaryotes to control proteins synthesis. Indeed, many translation factors are phosphoproteins whose
function can be regulated by extracellular signals. We will describe here the mechanism of translation initiation in
eukaryotes, with a particular emphasis on translation factors and their function.

37) How do you measure fidelity? (C212.2) (PO1,2,3,4)

Vendors use a variety of different methods to determine the fidelity of their DNA polymerases. One assay, first described by
Thomas Kunkel, uses portions of the lacZα gene in M13 bacteriophage to correlate host bacterial colony color changes with
errors in DNA synthesis. Building on the Kunkel assay, Wayne Barnes’ assay is a common permutation found in labs, in
which PCR is used to copy the entire lacZ gene and portions of two drug-resistance genes, with subsequent ligation, cloning,
transformation and blue/white-colony color determination. The readout of both assays is a white-colony phenotype caused by
the disruption of β-galactosidase activity that results from errors in the lacZ gene.
G. Gomathi
38) Write a short note on 70s ribosome. (C212.3) (PO1,2,3,4)
Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. Their small subunit
has a 16SRNA subunit (consisting of 1540 nucleotides) bound to 21 proteins. The large subunit is composed of
a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins.[

38) Explain the structure of RNA. (C212.3) (PO1,2,3,4)

RNA is typically single stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A
ribonucleotide in the RNA chain contains ribose (the pentose sugar),

one of the four nitrogenous bases (A, U, G, and C), and a phosphate group. The subtle structural difference between
the sugars gives DNA added stability, making DNA more suitable for storage of genetic information, whereas the
relative instability of RNA makes it more suitable for its more short-term functions. The RNA-specific
pyrimidine uracil forms a complementary base pair with adenine and is used instead of the thymine used in DNA.
Even though RNA is single stranded, most types of RNA molecules show extensive intramolecular base pairing
between complementary sequences within the RNA strand, creating a predictable three-dimensional structure
essential for their function (Figure 1 and Figure 2).

39) Outline the basic principles involved in eukaryotic RNA synthesis. (C212.3) (PO1,2,3,4)
Transcription between promoter (start) and termination (stop) signals, multi-subunit
polymerases in factories, the basic steps of transcription, initiation of synthesis of new chains,
synthesis 5'-to-3'.

40) What is the role of RNA polymerase? (C212.3) (PO1,2,3,4)

RNAP locally opens the double-stranded DNA (usually about four turns of the double helix) so that one strand of
the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription

41) Write the short note on structure of ribosome. (C212.3) (PO1,2,3,4)

 2 subunits
 50S and 30S in prokaryotes (70S)
 60S and 40S in eukaryotes (80S)
 In dynamic equilibrium
 Association- Mg2+ dependent in vitro
 In vivo cycle depends on protein factors

G. Gomathi
42) Distinguish between 70s and 80s ribosome. (C212.3) (PO1,2,3,4)
Difference between 80S and 70S Ribosomes:-

80S ribosome

1. They occur only in eukaryotic cells.

2. They occur inside the cytoplasm of eukaryotes either freely or attached to ER.

3. The ribosomes are larger in size with a length of (300—340 A) and breadth (200—240 A).

4. The sedimentation co-efficient is 80.

5. They are comparatively heavier, 4.0—4.5 million Daltons.

6. The two subunits are 40S and 60S.

7. The rRNAs of BOS ribosomes are 28S + 5.8S + 5S in larger subunit and 18S in smaller subunit.

8. The ribosomes possess less of rRNA as compared to protein (40: 60).

9. 80S ribosomes are synthesized inside the nucleolus.

10. It contains about 73 protein molecules, 40 in larger subunit and 33 in smaller subunit.

11. Protein synthesis is not inhibited by common antibiotics like chloramphenicol.

70S Ribosomes:

1. 70S ribosomes are found both in prokaryotes and eukaryotes.

G. Gomathi
2. The ribosomes are found freely inside the cytoplasm of prokaryotes and matrix of plastids and mitochondria of
eukaryotes.
3. They are comparatively smaller with a length of (200—290 A) and a diameter of (170— 210 A).

4. The sedimentation coefficient is 70.

5. 70S ribosomes are comparatively lighter, 2.7—3.0 million daltons.

6. The two subunits are 30S and 50S.

7. The rRNAs of 70S ribosomes are 23S + 5S (larger subunit) and 16S (smaller subunit).

8. The ribosomes contain more of rRNA than protein (60:40).

9. 70S ribosomes are synthesised in the cytoplasm of prokaryotes and matrix of semi-autonomous cell organelles.

10. It possesses about 55 protein molecules, 34 in larger subunit and 21 in smaller subunit.

11. Protein synthesis is inhibited by antibiotics like chloramphenicol.

43) How protein elongation take place in translation? (C212.3) (PO1,2,3,4)

Translation elongation is simply the ribosome travelling down the message, reading codons and bringing in the
proper aminoacyl tRNA’s to translate the message out to protein. The incoming aminoacyl tRNA is brought into
the ribosome A site, where it is matched with the codon being presented. Once it has been secured (by hydrolysis of
GTP to “fix” it in place), the peptidyltransferasereaction occurs. This is where the bond between the peptide and
the aminoacyl tRNA in the P site is broken, while a new bond is simultaneously formed between the (momentarily
unattached) peptide and the new amino acid in the A site. The ribosome then moves over by 3 bases, the spent
tRNA is ejected from the E site.

44) Describe the process of translation termination? (C212.3) (PO1,2,3,4)

Termination of translation occurs when the ribosome encounters a stop codon. There are slighty different views as
to what happens; some textbooks state that there is a release factor bound to the stop codon, that displaces the
ribosome when it reaches that point. Dr. Webb prefers the idea that the stop codon is simply a codon that has no
matching tRNA, so the ribosome stalls, waiting for the next aminoacyl tRNA. This stall causes the ribosome to
destabilize, and release factors come in to disassemble the ribosome and cut free the peptide strand.

45) Write a note on activation of amino acids. (C212.3) (PO1,2,3,4)

Amino acid activation refers to the attachment of an amino acid to its Transfer RNA (tRNA).
 Aminoacyl transferase binds Adenosine triphosphate (ATP) to amino acid, PP is released.
 Aminoacyl transferase binds AMP-amino acid to tRNA. The AMP is used in this step.

During amino acid activation the amino acids (aa) are attached to their corresponding tRNA. [1] The coupling
reactions are catalysed by a group of enzymes called aminoacyl-tRNA synthetases (named after the reaction
product aminoacyl-tRNA or aa-tRNA). The coupling reaction proceeds in two steps:
1. aa + ATP ⟶ aa-AMP + PP, (pyrophosphate) G. Gomathi
2. aa-AMP + tRNA ⟶ aa-tRNA + AMP
46) Explain the role of IF1(C212.3) (PO1,2,3,4)
Prokaryotic initiation factor-1 associates with the 30S ribosomal subunit in the A site and prevents an aminoacyl-
tRNA from entering. It modulates IF2 binding to the ribosome by increasing its affinity. It may also prevent the
50S subunit from binding, stopping the formation of the 70S subunit. It also contains a β-domain fold common for
nucleic acid binding proteins.

47) Explain the role of IF2(C212.3) (PO1,2,3,4)

Prokaryotic initiation factor-2 binds to an initiator tRNA and controls the entry of that tRNA into the ribosome. IF2,
bound to GTP, binds to the 30S P site. After associating with the 30S subunit, fMet-tRNA f binds to the IF2 then IF2
transfers the tRNA into the partial P site. When the 50S subunit joins, it hydrolyzes GTP to GDP and P i, causing a
conformational change in the IF2 that causes IF2 to release and allow the 70S subunit to form.

48) Explain the role of IF 3(C212.3) (PO1,2,3,4)

Prokaryotic initiation factor-3 is not universally found in all bacterial species but in E. coli it is required for the 30S
subunit to bind to the initiation site in mRNA. In addition, it has several other jobs including stabilization of free
30S subunits, facilitation of 30S subunits binding to mRNA and checking for accuracy against the first aminoacyl-
tRNA. It also allows for rapid codon-anticodon pairing for the initiator tRNA to bind quickly to. IF3 is required by
the small subunit to form initiation complexes, but has to be released to allow the 50S subunit to bind.

49) Explain the role of EF Tu (C212.3) (PO1,2,3,4)

EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor responsible for catalyzing the
binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and
binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one
of the most abundant and highly conserved proteins in prokaryotes.

50) Explain the role of EF Ts(C212.3) (PO1,2,3,4)

EF-Ts (elongation factor thermo stable) is one of the prokaryotic elongation factors.
EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu (elongation factor thermo unstable), catalyzing
the release of guanosine diphosphate from EF-Tu. This enables EF-Tu to bind to a new guanosine
triphosphate molecule, release EF-Ts, and go on to catalyze another aminoacyl tRNA addition.

UNIT IV – TRANSLATION
1. Why codon degeneracy is important to the cells? (May 2019)
Codon degeneracy refers to a single amino acid being encoded by more than one codon.
Traditionally, there are total 20 amino acids which code for a wide vareity of proteins in a living
organism. Nucleotides are 4. A codon in made of 3 nucleotides. Through simple maths it can be
determined that total number of different colons will be 64. One is a start codon, 3 are stop codons
and rest code for the 20 amino acids. This code is universal except in a few cases like the
mitochondria.

It has been suggested that the degeneracy makes the DNA more tolerant to point mutations. It isn't
G. Gomathi
necessary a point mutation in a codon will lead to change in conformation of the peptide. It might be
replaced by a synonymous amino acid. Thereby no change in the final protein is seen.
2. What is the role of amino acyl t-RNA synthetases in protein biosynthesis? (May 2019)

An aminoacyl-tRNA synthetase (aaRS or ARS), also called tRNA-ligase, is an enzyme that

attaches the appropriate amino acid onto its tRNA. It does so by catalyzing the esterification of a
specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form
an aminoacyl-tRNA. In humans, the 20 different types of aa-tRNA are made by the 20 different
aminoacyl-tRNA synthetases, one for each amino acid of the genetic code.
This is sometimes called "charging" or "loading" the tRNA with the amino acid. Once the tRNA is
charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to
the genetic code. Aminoacyl tRNA therefore plays an important role in RNA translation, the
expression of genes to create proteins.

3. Explain DNA foot printing. (C212.4) (PO1,2,3,4)

DNA footprinting is a method of investigating the sequence specificity of DNA-
binding proteins in vitro. This technique can be used to study protein-DNA interactions both
outside and within cells. The regulation of transcription has been studied extensively, and yet
there is still much that is not known. Transcription factors and associated proteins that bind
promoters, enhancers, or silencers to drive or repress transcription are fundamental to
understanding the unique regulation of individual genes within the genome. Techniques like
DNA footprinting will help elucidate which proteins bind to these regions of DNA and
unravel the complexities of transcriptional control.

4. Write a note on types of RNA splicing. (C212.4) (PO1,2,3,4)

In molecular biology and genetics, splicing is a modification of the nascent pre-

mRNA taking place after or concurrently with its transcription, in which introns are removed
and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can
be used to produce a correct protein through translation. For many eukaryotic introns,
splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of
small nuclear ribonucleoproteins (snRNPs), but there are also self-splicing introns.

5. What is conditional mutant? (C212.4) (PO1,2,3,4)

Mutation that has the wild-type phenotype under certain (permissive) environmental
conditions and a mutant phenotype under other (restrictive) conditions

6. What is the reading frame of an mRna? (C212.4) (PO1,2,3,4)

A reading frame is a way of breaking the sequence of nucleotides in a nucleic acid

such as a DNA or RNA into a set of consecutive triplets, called codons. When read as triplets,
a nucleic acid molecule may in general have six reading frames, three reading in one
direction along one strand and three reading in the other direction along the complementary
strand. In general, only one reading frame in a given section of a nucleic acid is biologically
relevant

7. Explain how hydrophobic interactions are important in stabilizing the DNA structure. (C212.4)
(PO1,2,3,4)
G. Gomathi
A noncovalent bond is a type of chemical bond, typically between macromolecules,
that does not involve the sharing of pairs of electrons, but rather involves more dispersed
variations of electromagnetic interactions. The noncovalent bond is the dominant type of
bond between supermolecules in supermolecular chemistry. Noncovalent bonds are critical in
maintaining the three-dimensional structure of large molecules, such as proteins and nucleic
acids, and are involved in many biological processes in which large molecules bind
specifically but transiently to one another. The energy released in the formation of
noncovalent bonds is on the order of 1-5 kcal per mol. There are four commonly mentioned
types of non-covalent interactions: hydrogen bonds, ionic bonds, van der Waals forces, and
hydrophobic interactions.

8. What is denaturation mapping? (C212.4) (PO1,2,3,4)

The identification of regions of low thermal (or alkali) stability (i.e., of high A+T content) in
a duplex DNA molecule, by trapping the partly melted structure and blocking renaturation,
e.g. with formaldehyde (which preferentially couples with the amino groups of the single-
stranded regions), and subsequently examining the specimen by electron microscopy.

9. Explain generalized transduction(C212.4) (PO1,2,3,4)

Transduction is a phenomenon in which bacterial DNA is transferred from one

bacterial cell to another by a phage particle. Phage particles that contain bacterial DNA are
called Transducing Particles. There are two types of transducing particles-generalized and
specialized.

10. What do you understand by cyclically permuted? (C212.4) (PO1,2,3,4)

A cyclic permutation or circular permutation is a permutation built from one or more

sets of elements in cyclic order.

11. Distinguish redundant and synonyms in genetic code(C212.4) (PO1,2,3,4)

The genetic code consists of 64 triplets of nucleotides. These triplets are called
codons. With three exceptions, each codon encodes for one of the 20 amino acids used in the
synthesis of proteins. That produces some redundancy in the code: most of the amino acids
being encoded by more than one codon

12. Define leaky mutation(C212.4) (PO1,2,3,4)

A mutant (typically an auxotroph) that results from a partial rather than a complete
inactivation of the wild-type function.

13. Define transcription process(C212.4) (PO1,2,3,4)

Flow of genetic information from DNA to the messenger RNA (mRNA) to express
the genome for biosynthesis of proteins. The transcription process is otherwise called as
synthesis of mRNA from DNA template either of the DNA strand by using enzyme RNA
polymerase.

14. Write short notes on prokaryotic RNA polymerases. (C212.4) (PO1,2,3,4)

G. Gomathi
RNA polymerase is the single enzyme present in the nucleus which is responsible for
the synthesis of all kinds of RNAs like mRNA, tRNA and rRNA. RNA polymerase present in
prokaryotic as well as eukaryotic cells is slightly differing in their structure and has many
subunits. This cluster of enzyme is responsible for different functions during the mRNA
synthesis or transcription process.

15. Mention briefly on sigma (σ) factor. (C212.4) (PO1,2,3,4)

The sigma subunits present in the RNA polymerase of prokaryotic cell helps in
recognition of start signals during mRNA synthesis. The sigma subunits are otherwise called
sigma factors, which directs RNA polymerase in selecting the initiation sites. Once the RNA
synthesis initiated, the sigma factors dissociate from the DNA and combine in the next
transcription cycle.

16. Write on promoter and terminator region. (C212.4) (PO1,2,3,4)

The promoter region or site is the special locations of DNA where the special region
contain specific nucleotides are present in which the RNA polymerase binds for initiation of
transcription process. This is otherwise called promoter site. The terminator region is the
special site of DNA, which contain specific nucleotide sequences, which are responsible for
terminating or stop the transcription process. This is otherwise called terminator region.

17. Write short notes on prokaryotic promoter(C212.4) (PO1,2,3,4)

The RNA polymerase binding site of the prokaryotic DNA region is called promoter
region or promoter site. This region contain 41 – 44 bp in E.coli. Promoter is the start site,
which contains 90% purine bases. The upstream from the promoter site is a 6 bp region
described as TATAAT sequence or TATAAT box. This is otherwise called as pribnow box.
This lies 10 bp region as – 10 sequence. I.e. –18 to –12 region. Another sequence is
TTGACA is lying –35 sequences on upstream is called recognition region. The typical
prokaryotic DNA use –35 and 10 sequences for transcription.

18. Describe pribnow box. (C212.4) (PO1,2,3,4)

The pribnow box is otherwise described as the promoter region of DNA. The
upstream from the promoter site is a 6 bp region described as TATAAT sequence or
TATAAT box. This is otherwise called as pribnow box. This lies 10 bp region as – 10
sequence. I.e. –18 to –12 region.

19. Where is consensus TATA sequence seen? Write the significance of it. (C212.4) (PO1,2,3,4)

The consensus TATA sequence is seen in the promoter region of prokaryotic DNA.
This is more helpful in recognition of RNA polymerase to bind for transcription process. This
consensus sequences occur at –10 bp of promoter region.

20. What is upstream and downstream site? (C212.4) (PO1,2,3,4)

The upstream and downstream sites are occurring at the transcription region of DNA.
The Upstream is the sequence region, which is prior to the start point of the promoter (from –
1 sequence). The Downstream site is the region after the start point of the promoter region
G. Gomathi
(from
+1 sequence).
21. What is promoter complex? (C212.4) (PO1,2,3,4)

The promoter complex is described as the combine product occurred after the
holoenzymes (RNA polymerase unit) binds at promoter site. When DNA is in double helix
stage, it is called closed promoter complex. Once the double helix is unwinded, then it is
called open promoter complex. After formation of promoter complex, the transcription state
is begin to start.

22. List out the unusual bases found in t RNA(C212.4) (PO1,2,3,4)

Unusual bases

tRNAs contain many unusual bases as shown by the illustrated side

chains: 1-methyl adenosine is a modified adenosine base found on

tRNA:

N2, N2-dimethylguanosine is a modified guanosine:

23. Describe few post translatory mechanisms(C212.4) (PO1,2,3,4)

Post-translational modifications can occur on the amino acid side chains or at the
protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino
acids by modifying an existing functional group or introducing a new one such as phosphate.
Phosphorylation is a very common mechanism for regulating the activity of enzymes and is
the most common post-translational modification . Many eukaryotic proteins also have
carbohydrate molecules attached to them in a process called glycosylation,G.which Gomathi
can
promote protein folding and improve stability as well as serving regulatory functions.
Attachment of lipid molecules, known as lipidation, often targets a protein or part of a protein
attached to the cell membrane.Other forms of post-translational modification consist of
cleaving peptide bonds, as in processing a propeptide to a mature form or removing the
initiator methionine residue. The formation of disulfide bonds from cysteine residues may
also be referred to as a post- translational modification.

24. How shine-dalgarno sequence initiate translation? (C212.4) (PO1,2,3,4)

G. Gomathi
In procaryotes, a ribosome with tRNA which carries methionine binds to the specific region
of mRNA and recognizes AUG codon nearby and protein synthesis begins. In this process the
main factors are ribosome, tRNA with methionine(fMet-tRNA-Metf), and mRNA. In
addition, at least initiation factors and GTP molecule are required to ensure the efficiency and
fidelity of this process.

Bacterial mRNAs are commonly polycistronic. That means that they encode multiple proteins
that are separatedly translated from the same mRNA molecule. Sometimes coding regions
overlap, but it may not affect the fidelity of translation. Sometimes coding regions overlap by
one base, which will be like UG [AUG].

In bacterial mRNA, ribosome binding site and start codon play important roles for translation
initiation. Ribosome binding site is where the 30S small subunit binds first on mRNA. This
site contains purine rich sequence which is called Shine-Dalgarno sequence. The 3' terminal
of 16S rRNA in 30S subunit binds to this sequence and helps 30S subunit to bind to mRNA.

25. Write down the phases of protein synthesis(C212.4) (PO1,2,3,4)

1. Activation of amino acids.

2. Initiation
3. Elongation
4. Termination
5. Folding and processing

26. Write short notes on Transpeptidation(C212.4) (PO1,2,3,4)

 The peptide bond is formed in the second stage of the elongation cycle
through the nucleophilic displacement of the P site tRNA by the amino group
of the 3’ linked aminoacyl tRNA in the A site.
 The nascent polypeptide chain is thereby lengthened at its C terminus by one
residue and transferred to the A site tRNA a process called transpeptidation.
 Peptidyl transferase activity probably appears on the 23s RNA of ribosome.
That can catalyse the reaction

27. Write short notes on Translocation(C212.4) (PO1,2,3,4)

 If in the final stage of elongation cycle, uncharged P site tRNA is transferred

to E site, it is former occupant having been previously expelled.
Simultaneously, in a process known as translocation, the peptidyl tRNA in the
A site, together with its bound mRNA is moved to the P site.
 The translocation process requires the participation of an elongation factor,
EF-G that binds to the ribosome together with GTP and is only released upon
hydrolysis of the GTP to GPP + Pi.
 EF-G release is pre requists for beginning the next elongation cycle because
the ribosomal binding sites of EF-G and EF-TU partially overlap and hence
their binding is mutually exclusive.

G. Gomathi
 28. Write short notes on start and stop codons(C212.4) (PO1,2,3,4)

In translation codons of three nucleotides determine which amino acid will be added
next in the growing protein chain. The start codon is usually AUG, while the stop codons are
UAA, UAG, and UGA. The open reading frame (ORF) is that portion of a DNA segment
which will putatively code for a protein; it begins with a start codon and ends with a stop
codon.

29. What are two types of codes? (C212.4) (PO1,2,3,4)

There are several ways in which a codon could be read from a mRNA molecule. The
two most important alternatives originally considered are the overlapping and nonoverlapping
codes. In an overlapping code each base serves as the first base of some codon; in a
nonoverlapping code, each base is used in only one codon.

30. Write short notes on Cycloheximide(C212.4) (PO1,2,3,4)

Cycloheximide is a chemical inhibitor of the peptidyl transferase complex of the 60S

subunit and hence inhibits formation of the peptide bond. Substances like Cycloheximide are
commonly used in cancer chemotherapy.

31. Write short notes on components of Translation process(C212.4) (PO1,2,3,4)

mRNA: – Eukaryotes: made in the nucleus, transported to the cytoplasm. –

Prokaryotes: transcription and translation occur concurrently.
tRNA: Adaptor molecules that mediate the transfer of information from nucleic acids to protein
Ribosomes: manufacturing units of a cell; located in the cytoplasm. Contain ribosomal RNA
and proteins.
Enzymes: required for the attachment of amino acids to the correct tRNA molecule, and
for peptide bond formation between amino acids.
Proteins: soluble factors necessary for proper initiation, elongation and
termination of translation.

32. When the ribosome encounters a stop codon? (C212.4) (PO1,2,3,4)

 There is no tRNA available to bind to the A site of the ribosome,

 Instead a release factor binds to it.

33. Define Nucleosomes. (C212.4) (PO1,2,3,4)

A nucleosome is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA

wound
in sequence around eight [1] histone protein cores.[2] This structure is often compared to thread
wrapped
around a spool

32) Explain Histones protein? (C212.4) (PO1,2,3,4) G. Gomathi

 In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package
and order the DNA into structural units called nucleosomes.[1][2] They are the chief protein
components of chromatin, acting as spools around which DNA winds, and playing a role
in gene regulation. Without histones, the unwound DNA in chromosomes would be very long
(a length to width ratio of more than 10 million to 1 in human DNA). For example, each
human diploid cell (containing 23 pairs of chromosomes) has about 1.8 meters of DNA; wound
on the histones, the diploid cell has about 90 micrometers (0.09 mm) of chromatin. When the
diploid cells are duplicated and condensed during mitosis, the result is about 120 micrometers
of chromosomes.[3]

33) Comment on concept of Gene. (C212.4) (PO1,2,3,4)

In biology, a gene is a sequence of DNA or RNA that codes for a molecule that has a function.
During gene expression, the DNA is first copied into RNA. The RNA can be directly
functional or be the intermediate template for a protein that performs a function. The
transmission of genes to an organism's offspring is the basis of the inheritance of phenotypic
traits. These genes make up different DNA sequences called genotypes. Genotypes along with
environmental and developmental factors determine what the phenotypes will be. Most
biological traits are under the influence of polygenes(many different genes) as well as gene–
environment interactions. Some genetic traits are instantly visible, such as eye color or number
of limbs, and some are not, such as blood type, risk for specific diseases, or the thousands of
basic biochemical processes that constitute life.

34) Define Intron. (C212.4) (PO1,2,3,4)

An intron is any nucleotide sequence within a gene that is removed by RNA

[1][2]
splicing during maturation of the final RNA product. The word intron is derived from the
term intragenic region, i.e. a region inside a gene.[3] The term intron refers to both the DNA
sequence within a gene and the corresponding sequence in RNA transcripts.[4] Sequences that
are joined together in the final mature RNA after RNA splicing are exons. Introns are found in
the genes of most organisms and many viruses, and can be located in a wide range of genes,
including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA).
When proteins are generated from intron-containing genes, RNA splicing takes place as part of
the RNA processing pathway that follows transcription and precedes translation.

35) Comment on Exon as an important part of m-RNA. (C212.4) (PO1,2,3,4)

An exon is any part of a gene that will encode a part of the final mature RNA produced by that
gene after introns have been removed by RNA splicing. The term exon refers to both the DNA
sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA
splicing, introns are removed and exons are covalently joined to one another as part of
generating the mature messenger RNA. Just as the entire set of genes for a species constitutes
the genome, the entire set of exons constitutes the exome.

36) Explain role of Non Histone proteins(C212.4) (PO1,2,3,4)

Histone gene expression was studied during the cell cycle of continuously dividing HeLa S3-
cells and following stimulation of confluent monolayers of WI-38 human diploid fibroblasts to
proliferate. The presence of histone messenger RNA (mRNA) sequences G. Gomathi
was assayed by
hybridization to a 3H-labeled single-stranded DNA complementary to histone mRNA's. In
HeLa S3-cells, histone mRNA sequences were found in the nucleus and associated with
 polyribosomes during S phase but not during G1. Transcripts of S-phase chromatin contained
histone mRNA sequences but those of G1 chromatin did not. Similarly, in WI-38 cells
association of histone mRNA sequences with polyribosomes and transcription of histone
mRNA sequences from chromatin parallel DNA replication. Together these results suggest that
the regulation of histone gene expression resides, at least in part, at the transcriptional level.
Chromatin reconstitution studies provide evidence that nonhistone chromosomal proteins play a
key role in activation of histone gene transcription during the period of the cell cycle when
DNA is replicated. Phosphate groups associated with the S-phase nonhistone chromosomal
proteins appear to be functionally involved in the control of histone gene readout. Although
WI-38 human diploid fibroblasts transformed by SV40 exhibit morphological and biochemical
modifications characteristic of neoplastic cells, transcription of histone mRNA sequences
remains unaltered.

37) Define – a) Chromatin (C212.4) (PO1,2,3,4)

Chromatin is a complex of DNA, RNA, and protein found in eukaryotic cells.[1] Its primary
function is packaging very long DNA molecules into a more compact, denser shape, which
prevents the strands from becoming tangled and plays important roles in reinforcing the DNA
during cell division, preventing DNA damage, and regulating gene expression and DNA
replication. During mitosis and meiosis, chromatin facilitates proper segregation of
the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this
stage are the result of DNA being coiled into highly condensed networks of chromatin.
b) Chromosome
In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called
chromosomes. Each chromosome is made up of DNA tightly coiled many times around
proteins called histones that support its structure.
Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the
cell is not dividing. However, the DNA that makes up chromosomes becomes more tightly
packed during cell division and is then visible under a microscope. Most of what researchers
know about chromosomes was learned by observing chromosomes during cell division.

38) Define Genetic code & Codon. (C212.4) (PO1,2,3,4)

39) Explain degeneracy of genetic code. (C212.4) (PO1,2,3,4)

40) Explain how Histine protein is important in DNA packaging? (C212.4) (PO1,2,3,4)

The double helix of DNA is highly negatively charged due to all the negatively charged
phosphates in the backbone. All that negative charge must be counterbalanced by a positive charge,
and the cell makes proteins called histones that bind DNA and aid in DNA's packaging. Histones are
positively charged proteins that wrap up DNA through interactions between their positive charges and
the negative charges of DNA. Double-stranded DNA loops around 8 histones twice, forming
the nucleosome, which is the building block of chromatin packaging.
41) What is conditional mutant? (C212.4) (PO1,2,3,4)

Mutation that has the wild-type phenotype under certain (permissive) environmental conditions and a
G. Gomathi
mutant phenotype under other (restrictive) conditions

43) Discuss nucleosomes as fundamentle unit of DNA organization(C212.4) (PO1,2,3,4)

 The fundamental unit of chromatin, termed the nucleosome, is composed of DNA and
histone proteins. This structure provides the first level of compaction of DNA into the nucleus.
Nucleosomes are regularly spaced along the genome to form a nucleofilament which can adopt
higher levels of compaction (Fig 1 and 3), ultimately resulting in the highly condensed metaphase
chromosome. Within an interphase nucleus chromatin is organized into functional territories..

44) Comment on organization of Chromatin(C212.4) (PO1,2,3,4)

Structural organization of chromatin 3.1 The nucleosome and “Beads on string” structure The
first level of packing involves the binding of the chromosomal DNA to histones. In eukaryotes,
DNA is tightly bound to form a repeating array of DNA-protein particles called nucleosomes.
Histones play a crucial role in packing this very long DNA molecule in an orderly way into
nucleus which is only a few micrometers in diameter. Thus, nucleosomes are the fundamental
packing unit particles of the chromatin and give chromatin a “beads on string” appearance in
the electron micrographs. Each nucleosome bead is separated from the next by a region of
linker DNA (Fig.3). There are five main types of histones called H1, H2a, H2b, H3 and H4.
Histones are very basic proteins, about 25% of their amino acids are lysine and arginine. So
histones have a large number of positively charged amino acid side chains. Their positively
charged groups therefore bind to the negatively charged phosphate groups of DNA.

45) Distinguish between Histone & Non histone proteins. (C212.4) (PO1,2,3,4)

Histone vs Nonhistone Proteins

Histone proteins are the principal Nonhistone proteins are components of
protein component of chromatin. chromatin.
Major Function
They act as spools for DNA to wind They act mainly as scaffolding proteins for
and become shorter in length. DNA.
Types
H1/H5, H2A, H2B, H3, and H4 are Scaffold proteins, Heterochromatin Protein 1,
types of histones. DNA polymerase, Polycomb, etc. are some types
of nonhistones.
Involvement of Nucleosome
Histone proteins are the core proteins Nonhistone proteins are not a part of a
of a nucleosome. nucleosome.
Conserved Sequence
Histone proteins are conserved across Nonhistone proteins are not conserved across
species. species.
Role in Gene Expression
Histone proteins are involved in gene Nonhistone proteins are not involved in gene
expression regulation expression regulation

46) Write on promoter and terminator region. (C212.4) (PO1,2,3,4)

G. Gomathi
The promoter region or site is the special locations of DNA where the special region
contain specific nucleotides are present in which the RNA polymerase binds for initiation of
 transcription process. This is otherwise called promoter site. The terminator region is the
special site of DNA, which contain specific nucleotide sequences, which are responsible for
terminating or stop the transcription process. This is otherwise called terminator region.

47. Write short notes on prokaryotic promoter(C212.4) (PO1,2,3,4)

The RNA polymerase binding site of the prokaryotic DNA region is called promoter
region or promoter site. This region contain 41 – 44 bp in E.coli. Promoter is the start site,
which contains 90% purine bases. The upstream from the promoter site is a 6 bp region
described as TATAAT sequence or TATAAT box. This is otherwise called as pribnow box.
This lies 10 bp region as – 10 sequence. I.e. –18 to –12 region. Another sequence is
TTGACA is lying –35 sequences on upstream is called recognition region. The typical
prokaryotic DNA use –35 and 10 sequences for transcription.

48. Describe pribnow box. (C212.4) (PO1,2,3,4)

The pribnow box is otherwise described as the promoter region of DNA. The
upstream from the promoter site is a 6 bp region described as TATAAT sequence or
TATAAT box. This is otherwise called as pribnow box. This lies 10 bp region as – 10
sequence. I.e. –18 to –12 region.

49. Where is consensus TATA sequence seen? Write the significance of it. (C212.4) (PO1,2,3,4)

The consensus TATA sequence is seen in the promoter region of prokaryotic DNA.
This is more helpful in recognition of RNA polymerase to bind for transcription process. This
consensus sequences occur at –10 bp of promoter region.

50. Define Chromosome

In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called
chromosomes. Each chromosome is made up of DNA tightly coiled many times around
proteins called histones that support its structure.

UNIT V - REGULATION OF GENE

EXPRESSION

1. What are the structural genes present in E.Coli trp operon? (May 2019)

Trp operon contains five structural genes: trpE, trpD, trpC, trpB, and trpA, which encode
enzymatic parts of the pathway. It also contains a repressive regulator gene called trpR.
trpR has a promoter where RNA polymerase binds and synthesizes mRNA for a
G. Gomathi
regulatory protein.

2. What are temperate phages? (May 2019)

 Temperate phages are bacteriophages that can choose between the lytic and the lysogenic
pathways of development. The lytic pathway is similar to that of virulent phages. In the
lysogenic pathway, the virus remains dormant until induction.

3. What is Gene organization? (C212.5) (PO1,2,3,4)

Genes inside the cell follow several layers of organisation to enable the long DNA to
be compacted into the chromosome fibers. In the first level of packing, DNA is wrapped
around 4 pairs of proteins called Histones in a “beads-on-string” fashion. These nucleosomes
then coil around each other in the form of a helix, with around 6 nucleosomes forming one
turn of a helix. These helices form the long chromatin fibers which, with series of turns and
loops, forms the third level of spatial DNA organisation. Finally, these chromatin fibers are
compactly packed inside the chromosome. In the Chromosome, the chromatin fibers are
wrapped around a protein scaffold.

4. How does gene regulation occur? (C212.5) (PO1,2,3,4)

A gene (or genetic) regulatory network (GRN) is a collection of molecular
regulators that interact with each other and with other substances in the cell to govern the
gene expression levels of mRNA and proteins. These play a central role in morphogenesis,
the creation of body structures, which in turn is central to evolutionary developmental
biology (evo-devo).
The regulator can be DNA, RNA, protein and complexes of these. The interaction can be
direct or indirect (through transcribed RNA or translated protein). In general, each mRNA
molecule goes on to make a specific protein (or set of proteins). In some cases this
protein will be structural, and will accumulate at the cell membrane or within the cell to give
it particular structural properties. In other cases the protein will be an enzyme, i.e., a micro-
machine that catalyses a certain reaction, such as the breakdown of a food source or toxin.
Some proteins though serve only to activate other genes, and these are the transcription
factors that are the main players in regulatory networks or cascades. By binding to the
promoter region at the start of other genes they turn them on, initiating the production of
another protein, and so on. Some transcription factors are inhibitory.

5. How many amino acids are present in a nascent polypeptide decoded from mRNA
with the reading frame having 1002 nucleotides? (C212.5) (PO1,2,3,4)
a. In molecular biology, a reading frame is a way of dividing the sequence of
nucleotides in a nucleic acid (DNAor RNA) molecule into a set of consecutive,
non- overlapping triplets. Where these triplets equate to amino acids or stop
signals during translation, they are called codons.
b. A single strand of a nucleic acid molecule has a phosphoryl end, called the 5′-end,
and a hydroxyl or 3′-end. These define the 5'→3' direction. There are three
reading frames that can be read in this 5'→3' direction, each beginning from a
different nucleotide in a triplet. In a double stranded nucleic acid, an additional
three reading frames may be read from the other, complementary strand in the
5'→3' direction along this strand. As the two strands of a double stranded nucleic
acid molecule are antiparallel, the 5'→3' direction on the second strand
corresponds to the 3'→5' direction along the first strand.

6. Why DNA replication is called semi-conservative? (C212.5) (PO1,2,3,4)

DNA replication is semi-conservative because each helix that is createdG. contains
Gomathione
strand from the helix from which it was copied. The replication of one helix results in two
daughter helices each of which contains one of the original parental helical strands. It is semi-
conservative because half of each parent helix is conserved in each daughter helix.

7. What are introns? (C212.5) (PO1,2,3,4)

An intron is a nucleotide sequence within a gene. It is a noncoding sequence. During
the final maturation of the RNA product, the RNA removes it by splicing.1 The term intron
pertains to the DNAsequence within a gene as well as the corresponding sequence
in the RNA transcripts.2 It is used in contrast to the nucleotide sequences joined together
in a mature RNA after splicing calledexons. Sometimes, the term intron is used
synonymously to intervening sequences. However, the latter is a broader term that includes
inteins and UTRs, apart from the introns. Introns occur in the genes of many organisms (e.g.
eukaryotes), including viruses. They are also present in the genes of mitochondria and
chloroplasts. Introns are believed to be essential in allowing rapid evolution of proteins
through exon shuffling.

8. Name the cell organelle where protein synthesis takes place(C212.5) (PO1,2,3,4)
The rough endoplasmic reticulum is where most protein synthesis occurs in the cell.
The function of the smooth endoplasmic reticulum is to synthesize lipids in the cell. The
smooth ER also helps in the detoxification of harmful substances in the cell. Ribosomes-
Organelles that help in the synthesis of proteins.

9. Give reason – why DNA is acidic in nature(C212.5) (PO1,2,3,4)

DNA is made of three types of molecules in equal proportions - basic nucleotides,
sugar deoxyribose and acidic phosphate groups. The bases are on the inside of the helix and
partly hidden from the outside. Deoxyrybose and phopshates are on the outside, forming a
backbone.
Though the proportions are equal, the nucleotides are weak bases, so the overall pH is acidic.

10. Name the process by which RNA is synthesised from DNA. (C212.5) (PO1,2,3,4)

Gene expression is the process by which the genetic code - the nucleotide sequence - of a gene is
used to direct protein synthesis and produce the structures of the cell. Genes that code for amino
acid sequences are known as 'structural genes'.
The process of gene expression involves two main stages:
Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase,
and the processing of the resulting mRNA molecule. Translation: the use of mRNA to direct
protein synthesis, and the subsequent post-translational processing of the protein molecule.
Some genes are responsible for the production of other forms of RNA that play a role in
translation, including transfer RNA (tRNA) and ribosomal RNA (rRNA).

11. Why lac operon switches off in the absence of Lactose in E.coli? (C212.5) (PO1,2,3,4)
The lac operon, an inducible operon, is a mechanism used by bacterial cells as an
economical means to restrict the expression of the structural genes necessary for metabolizing
lactose, a disaccharide. These structural genes break down lactose when lactose is the best
carbon source available within its environs. E.coli utilizes the lac operon as a means of
controlling the expression of its lac genes in response to its environment. The primary carbon
source of this bacterium is glucose because it does not require a large amount of
energy to G. Gomathi
metabolize. It is a more efficient source of energy than lactose. In the presence of both
glucose and lactose, the bacterium will chose to metabolize glucose.
12. Why Chargaff’s rule is not applicable for RNA? (C212.5) (PO1,2,3,4)
RNA is found as a single stranded molecule. Chargaff's rule states that DNA helices
contain equal molar ratios of A to T and G to C. This is because DNA is found as a double
stranded helix in which A and T and G and C bases pair complementarily. RNA only forms
local helices meaning that it doesn't necessarily contain equal ratios.

13. Why the nucleotide ratio in RNA is not usually constant? (C212.5) (PO1,2,3,4)
Due to the absence of complementary base pairing. RNA is single stranded. So, the
nucleotide ratio is not constant in RNA.

14. Why is processed mRNA in eukaryotes is shorter than its gene? (C212.5) (PO1,2,3,4)
Processed mRNA in eukaryotes is shorter than its gene because the eukaryotic gene is
split gene and the transcribed mRNA has intron portions.

15. Name the process of RNA directed DNA synthesis(C212.5) (PO1,2,3,4)

A reverse transcriptase (RT) is an enzyme used to generate complementary
DNA (cDNA) from an RNA template, a process termedreverse transcription. It is mainly
associated with retroviruses. However, non-retroviruses also use RT (for example, the
hepatitis B virus, a member of the Hepadnaviridae, which are dsDNA-RT viruses, while
retroviruses are ssRNA viruses). RT inhibitors are widely used as antiretroviral drugs. RT
activities are also associated with the replication of chromosome ends (telomerase) and some
mobile genetic elements (retrotransposons).
Retroviral RT has three sequential biochemical activities:
(a) RNA-dependent DNA polymerase activity,
(b) ribonuclease H, and
(c) DNA-dependent DNA polymerase activity.
These activities are used by the retrovirus to convert single-stranded genomic RNA into
double- stranded cDNA which can integrate into the host genome, potentially generating a
long-term infection that can be very difficult to eradicate. The same sequence of reactions is
widely used in the laboratory to convert RNA to DNA for use in molecular cloning,
RNA sequencing, polymerase chain reaction (PCR), or genome analysis

16. Why codons are redundant? (C212.5) (PO1,2,3,4)

Degeneracy of codons is the redundancy of the genetic code, exhibited as the
multiplicity of three-base pair codon combinations that specify an amino acid. The
degeneracy of the genetic code is what accounts for the existence of synonymous mutations.
Degeneracy of the genetic code was identified by Lagerkvist. For instance, codons GAA and
GAG both specify glutamic acid and exhibit redundancy; but, neither specifies any other
amino acid and thus are not ambiguous or demonstrate no ambiguity. The codons encoding
one amino acid may differ in any of their three positions; however, more often than not, this
difference is in the second or third position. For instance, the amino acid glutamic acid is
specified by GAA and GAG codons (difference in the third position); the amino acid leucine
is specified by UUA, UUG, CUU, CUC, CUA, CUG codons (difference in the first or third
position); and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC
(difference in the first, second, or third position). Degeneracy results because there are more
codons than encodable amino acids. For example, if there were two bases per codon, then
only 16 amino acids could be coded for (4²=16). Because at least 21 codes are required (20
G.64Gomathi
amino acids plus stop) and the next largest number of bases is three, then 4³ gives possible
codons, meaning that some degeneracy must exist.
17. Why codons are sensible? (C212.5) (PO1,2,3,4)
The gene is represented by the sequences of bases in the DNA molecule, which can,
in a sense, be thought of as a "storage molecule" for genetic information. DNA is extremely
stable, a property critical to the maintenance of the integrity of the gene. This stability is
evidenced by the fact that DNA has been extracted from Egyptian mummies and extinct
animals such as the woolly mammoth. It can be extracted from dried blood or from a single
hair at a crime scene. Each cell contains a complete set of genes, but only certain of these
genes are active or "expressed" at any one time. When a gene is active, a "disposable" copy is
transcribed from the gene into codons contained in a messenger RNA (mRNA) molecule.
Unlike the DNA molecule, the mRNA molecule is relatively unstable and short-lived. This is
so that when a gene is turned off, the mRNA does not remain in the cell forever, running off
more proteins on the ribosomes that are no longer needed by the cell.

18. Why redundancy concept of genetic code does not apply to all amino acids? (C212.5)
(PO1,2,3,4)
The genetic code is said to be redundant in that the same amino acid residue can be
encoded by multiple, so-called synonymous, codons. If all properties of synonymous codons
were entirely equivalent, one would expect that they would be equally distributed along
protein coding sequences. However, many studies over the last three decades have
demonstrated that their distribution is not entirely random. It has been postulated that certain
codons may be translated by the ribosome faster than others and thus their non-random
distribution dictates how fast the ribosome moves along particular segments of the mRNA.
The reasons behind such segmental variability in the rates of protein synthesis, and thus
polypeptide emergence from the ribosome, have been explored by theoretical and
experimental approaches. Predictions of the relative rates at which particular codons are
translated and their impact on the nascent chain have not arrived at unequivocal conclusions.
This is probably due, at least in part, to variation in the basis for classification of codons as
“fast” or “slow”, as well as variability in the number and types of genes and proteins
analyzed. Recent methodological advances have allowed nucleotide- resolution studies of
ribosome residency times in entire transcriptomes, which confirm the non- uniform
movement of ribosomes along mRNAs and shed light on the actual determinants of rate
control. Moreover, experiments have begun to emerge that systematically examine the
influence of variations in ribosomal movement and the fate of the emerging polypeptide
chain.

19. Explain wobble hypothesis. (C212.5) (PO1,2,3,4)

Even before the genetic code had been elucidated, Francis Crick postulated that base
pairing of the mRNA codons with the tRNA anticodons would require precision in the first
two nucleotide positions but not so in the third position (the precise conformation of base
pairs , which refers to the hydrogen bonding between A-T (A-U in RNA) and C-G pairs is
known as Watson-Crick base pairing). The third position, in general, would need to be only
a purine (A or
G) or a pyrimidine (C or U). Crick called this phenomenon "wobble." This less-than-precise base
pairing would require fewer tRNA species. For example, tRNAGlucould pair with either GAA or GAG
codons. In looking at the codon table, one can see that, for the most part, the first two letters are
important to specify the particular amino acid. The only exceptions are AUG (Met) and UGG (Trp)
which, as indicated above, have only one codon each.

20. What are all the exceptions to the universal genetic code? (C212.5) G. Gomathi mea
ning
(PO1,2,3,4)
Usual
Organism Normal codon
New meaning
Mammalian AGA, AGG Arginine Stop codon
Mitochondria AUA Isoleucine Methionine
UGA Stop codon Tryptophan
Drosophila AGA, AGG Arginine Serine
Mitochondria AUA Isoleucine Methionine
UGA Stop codon Tryptophan
Yeast AUA Isoleucine Methionine
Mitochondria UGA Stop codon Tryptophan
CUA, CUC, CUG,
CUU Leucine Threonine
Higher plant UGA Stop codon Tryptophan
Mitochondria CGG Arginine Tryptophan
Protozoan nuclei UAA, UAG Stop codons Glutamine
Mycoplasma capricolum
bacteria UGA Stop codon Tryptophan

G. Gomathi
21. Explain Lysogeny. (C212.5) (PO1,2,3,4)
Lysogeny, or the lysogenic cycle, is one of two cycles of viral
reproduction (the lytic cycle is the other). Lysogeny is characterized by
integration of the bacteriophage nucleic acid into the host bacterium's
genome or formations of a circular replicon in the bacterium's
cytoplasm. In this condition the bacterium continues to live and
reproduce normally. The genetic material of the bacteriophage, called
aprophage, can be transmitted to daughter cells at each subsequent cell
division, and a later event (such as UV radiation or the presence of
certain chemicals) can release it, causing proliferation of new phages
via the lytic cycle. Lysogenic cycles can also occur ineukaryotes,
although the method of DNA incorporation is not fully understood. The
distinction between lysogenic and lytic cycles is that the spread of the
viral DNA occurs through the usual prokaryotic reproduction, while the
lytic phage is spread through the production of thousands of individual
phages capable of surviving and infecting other cells. The key
difference between the lytic cycle and the lysogenic cycle is that the
lysogenic cycle does not lyse the host cell.[2] Phages that replicate only
via the lytic cycle are known as virulent phages while phages that
replicate using both lytic and lysogenic cycles are known
astemperate phages. In the lysogenic cycle, the phage DNA first
integrates into the bacterial chromosome to produce the prophage.
When the bacterium reproduces, the prophage is also copied and is
present in each of the daughter cells. The daughter cells can continue to
replicate with the prophage present or the prophage can exit the
bacterial chromosome to initiate the lytic cycle.

22. Give the importance of leader sequence(C212.5) (PO1,2,3,4)

Some operons are under attenuator control, in which
transcription is initiated but is halted before the mRNA is transcribed.
This introductory region of the mRNA is called the leader sequence; it
includes the attenuator region, which can fold back on itself, forming a
stem-and- loop structure that blocks the RNA polymerase from
advancing along the DNA.

23. Allolactose control of lac operon. Explain(C212.5) (PO1,2,3,4)

The operon is under the control of the adjacent lacI gene,
encoding the lactose repressor. The repressor is a regulatory gene. In
the absence of allolactose, the inducer of the lac operon, the repressor
tetramer binds to the lac operator (lacO) and prevents RNA polymerase
from transcribing the operon.

24. Differentiate between positive and negative control mechanisms in

bacteria. (C212.5) (PO1,2,3,4)
Positive: binding of an activator (e.g., cAMP) to a DNA-binding
protein (e.g., CAP) stimulates that latter's DNA binding, and so
initiation by RNA polymerase.
Negative: binding of a repressor (e.g., the trp repressor) promotes
DNA binding, preventing initiation.
 25. Outline the effects that occur when tryptophan switches off
expression of the trp operon. (C212.5) (PO1,2,3,4)
Two molecules of tryptophan bind to the trp repressor (a helix-
turn-helix homodimer), increasing its affinity for the operator; the two
recognition helices tilt and enter the major groove about 10 bp apart to
contact the edge of the relevant bases. Now, the bound complex
prevents the template from attaching to the polymerase. When
tryptophan is absent, the now-unoccupied repressor dissociates from
the operator so the template can attach productively to the polymerase
(and the operon is transcribed).

26. How does the catabolite activator protein promote expression

of catabolic enzymes in bacteria? (C212.5) (PO1,2,3,4)
Falling glucose levels increase the concentration of cAMP,
which binds to CAP (a homodimer of a polypeptide containing a helix-
turn-helix motif), promoting its affinity for target sequences embedded
in the promoters of many genes encoding catabolic enzymes. Binding
in the major groove narrows it, while the opposing minor groove
widens; the result is a 40 kink. Now promoters attach more efficiently
to polymerases, so catabolic enzymes are expressed at higher levels.

27. How was 'Dolly' the lamb cloned? (C212.5) (PO1,2,3,4)

Dolly's genes are derived from the udder of a (white-faced) Finn Dorset
ewe. Cells from this ewe were arrested by serum starvation in G0, fused (using an electrical
pulse) with an enucleate egg (from a Scottish Blackface ewe) in meiotic metaphase II, and
the reconstituted egg cultured in the ligated oviduct of a (Scottish Blackface) foster mother
for 6 days. Then, the egg was recovered, checked to see that it had developed into a
blastocyst, reimplanted into a second (Scottish Blackface) foster mother, and allowed to
develop to term (giving white-faced Dolly). Dolly's nuclear genes (excepting any mutant
genes) are identical to those of her genetic mother (but not her foster mother).

28. By what mechanisms might selective gene expression be achieved?

(C212.5) (PO1,2,3,4)
E.g., DNA rearrangement (immunoglobulin genes) or
amplification (rDNA genes) or loss (Dipteran embryos) or
modification (methylation), alteration in the rate of transcriptional
initiation (many genes) or elongation (heat-shock locus), transcript
rearrangement (alternative splicing) or processing or degradation
(histone mRNA), differential transport of mRNA out of the nucleus or
to different locations (-actin), differential translation of mRNA,
differential protein degradation or stabilization.

29. Give an example of an experiment showing that differential

expression of a gene can require continuous regulation?
(C212.5) (PO1,2,3,4)
A typical muscle cell expresses the cell-adhesion molecule, N-
CAM, on its surface. The stability of the switches involved in
maintaining N-CAM expression were analyzed by fusing a mouse
myoblast with a human lung fibroblast (which does not express any
 muscle-specific proteins); the resulting heterokaryon expressed N-
CAM of both species. This suggests that a switch acting continuously
on the muscle nucleus can also switch on N-CAM in the human
nucleus.

30. Illustrate how the activity of rDNA genes can be inherited through
mitosis. (C212.5) (PO1,2,3,4)
NORs, tandem repeats of 45S rRNA genes carried on five pairs
of human chromosomes with perhaps only 6 loci being transcribed,
activity associated with UBF on mitotic chromosomes, those NORs
carrying UBF tend for form nucleoli in daughters.

31. How would you demonstrate the effects of a maternal effect gene in
Drosophila? (C212.5) (PO1,2,3,4)
Cross the (grandparent) fly carrying the mutant maternal-effect
allele with another fly carrying the same mutation (i.e., -/+ x -/+).
Although one-quarter of the resulting fertilized eggs are genotypically
-/-, they contain maternal products of the + gene, so the embryonic
body plan is laid down normally. When such embryos reach adulthood
they can be crossed with a wild-type male (i.e., -/- x +/+); then, the
resulting fertilized eggs have +/- genes in a cytoplasm that lacks any +
products from the mother. As a result, an apparently normal mother
lays an egg that then develops abnormally.

31) Give the role of three enzyme of Lac-operon(C212.5) (PO1,2,3,4)

The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide,
into glucose and galactose. lacY encodes Beta-galactoside permease, a membrane
protein which becomes embedded in the cytoplasmic membraneto enable the cellular
transport of lactose into the cell. Finally, lacA encodes Galactoside acetyltransferase.

32) Comment on importance of cAMP in Lac operon. (C212.5) (PO1,2,3,4)

cAMP is important because it controls the activity of an activator protein called CAP. When
CAP is bound to cAMP, it binds to a binding site on the lac operon thereby, activating
transcription[2].

33) What are structural genes? Explain their role. (C212.5) (PO1,2,3,4)
The three structural genes are: lacZ, lacY, and lacA.
 lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves
the disaccharide lactose into glucose and galactose.
 lacY encodes Beta-galactoside permease (LacY), a
transmembrane symporter that pumps β-galactosides including lactose into
the cell using a proton gradient in the same direction. Permease increases the
permeability of the cell to β-galactosides.
 lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers
an acetyl group from acetyl-CoA to β-galactosides.

Only lacZ and lacY appear to be necessary for lactose catabolism.

34) Define Promoter & Operator. (C212.5) (PO1,2,3,4)
Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is
recognized by RNA polymerase, which then initiates transcription. In RNA synthesis,
promoters indicate which genes should be used for messenger RNA creation – and, by
extension, control which proteins the cell produces.
Operator – a segment of DNA that a repressor binds to. It is classically defined in the lac
operon as a segment between the promoter and the genes of the operon. [14] The main
operator (O1) in the lac operon is located slightly downstream of the promoter; two
additional operators, O1 and O3 are located at -82 and +412, respectively. In the case of a
repressor, the repressor protein physically obstructs the RNA polymerase from
transcribing the genes

35) What is operon? Explain its concept briefly. (C212.5) (PO1,2,3,4)

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the
control of a single promoter.[1] The genes are transcribed together into an mRNA strand and
either translated together in the cytoplasm, or undergo splicing to
create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that
each encode a single gene product. The result of this is that the genes contained in the operon
are either expressed together or not at all. Several genes must be co-transcribed to define an
operon.

36) Compare positive & negative regulation. (C212.5) (PO1,2,3,4)

In negative regulation a repressor protein binds to an operator to prevent a gene from being
expressed.
In positive regulation a transcription factor is required to bind at the promoter in order to
enable RNA polymerase to initiate transcription.

37) Give the role of allolactose in lac operon. (C212.5) (PO1,2,3,4)

The inducer, allolactose, binds to the repressor subunits, preventing their assembly into an
active tetramer. Allolactose is produced from lactose by β-galactosidase at a steady low rate
and thus serves as a lactose signal.

38) Give the importance of regulatory genes. (C212.5) (PO1,2,3,4)

A regulator gene, regulator, or regulatory gene is a gene involved in controlling
the expression of one or more other genes. Regulatory sequences, which encode regulatory
genes, are often 5' to the start site of transcription of the gene they regulate. In addition, these
sequences can also be found 3' to the transcription start site. In both cases, whether the
regulatory sequence occurs before (5') or after (3') the gene it regulates, the sequence is often
many kilobases away from the transcription start site. A regulator gene may encode a protein,
or it may work at the level of RNA, as in the case of genes encoding microRNAs. An
example of a regulator gene is a gene that codes for a repressor protein that inhibits the
activity of an operator gene (a gene which binds repressor proteins thus inhibiting
the translation of RNA to protein via RNA polymerase).

39) What is junk DNA(C212.5) (PO1,2,3,4)

All eukaryotic cells posses more DNA than prokaryotic cells. A careful analysis of eukaryotic
DNA content reveals that the number of expressed genes is about 2 to 10 times more than
that of prokaryotes. But the total amount of DNA in eukaryotes is still much higher than in a
prokaryotic cell. This implies that there is a huge excess of DNA in eukaryotic cells that do
not code for any gene. This unused and unwanted DNA is reffered to as junk DNA.

40) Define polycistronic mRNA. (C212.5) (PO1,2,3,4)

Polycistronic mRNA refers to a messenger RNA which encodes two or more
proteins. Polycistronic messenger RNAs participates in the process of protein
synthesis (translation) in prokaryotes.
Many bacterial mRNAs are fully functional in their primary composition transcripts
meaning that they do not require any posttranscriptional modifications. In
prokaryotes an mRNA molecule usually contains multiple open reading frames
(ORFs) each corresponding to a single gene transcript – (polycistronic mRNA, also
the “polygenic mRNA” synonym is used) or, more rarely, the transcript of a single
gene (monocistronic or monogenic mRNA). In polycistronic mRNAs the
information for each ORF is translated into a polypeptide

41) Write notes on ‘Gene Dosage’(C212.5) (PO1,2,3,4)

Some gene products are required in much larger quantities than ohers. A common method of
attaining such large quantities of a particular gene product is by gene dosage. For example if
two genes X and Y are transcribed at the same rate and the translation efficiencies are also
same the amount of the products, A and B formed will be equal. If there are 20 copies of gene
X 20 times of product A would have been formed than product B. The histone family sets an
example to gene dosage effect. In order to synthesize huge amount of histone required to
form chromatin most cells contain hundreds of times of histone genes. This is known as gene
dosage.

42) Comment on importance of cAMP in Lac operon. (C212.5) (PO1,2,3,4)

cAMP is important because it controls the activity of an activator protein called CAP. When
CAP is bound to cAMP, it binds to a binding site on the lac operon thereby, activating
transcription[2].

43) Describe allolactose in lac operon. (C212.5) (PO1,2,3,4)

The inducer, allolactose, binds to the repressor subunits, preventing their assembly into an
active tetramer. Allolactose is produced from lactose by β-galactosidase at a steady low rate
and thus serves as a lactose signal.

44) Comment on Lac repressor protein. (C212.5) (PO1,2,3,4)

The lac repressor is a DNA-binding protein which inhibits the expression of genes coding
for proteins involved in the metabolism of lactose in bacteria. These genes are repressed
when lactose is not available to the cell, ensuring that the bacterium only invests energy in
the production of machinery necessary for uptake and utilization of lactose when lactose is
present. When lactose becomes available, it is converted into allolactose, which inhibits
the lac repressor's DNA binding ability. Loss of DNA binding by the lac repressor is required
for transcriptional activation of the operon.

45) Name the cell organelle where protein synthesis takes place(C212.5) (PO1,2,3,4)
 The rough endoplasmic reticulum is where most protein
synthesis occurs in the cell. The function of the smooth endoplasmic
reticulum is to synthesize lipids in the cell. The smooth ER also helps
in the detoxification of harmful substances in the cell. Ribosomes-
Organelles that help in the synthesis of proteins.

46)Give reason – why DNA is acidic in nature(C212.5) (PO1,2,3,4)

DNA is made of three types of molecules in equal proportions -
basic nucleotides, sugar deoxyribose and acidic phosphate groups. The
bases are on the inside of the helix and partly hidden from the outside.
Deoxyrybose and phopshates are on the outside, forming a backbone.
Though the proportions are equal, the nucleotides are weak bases, so the overall
pH is acidic.
47) Poly cistronic vs monocistronic mRNA

48) Give significance of an Inducer in Lac operon. (C212.5) (PO1,2,3,4)

In molecular biology, an inducer is a molecule that regulates gene expression.[1] An inducer
can bind to protein repressors or activators.
Inducers function by disabling repressors. The gene is expressed because an inducer binds to
the repressor. The binding of the inducer to the repressor prevents the repressor from binding
to the operator. RNA polymerase can then begin to transcribe operon genes.
Inducers also function by binding to activators. Activators generally bind poorly to activator
DNA sequences unless an inducer is present. Activator binds to an inducer and the complex
binds to the activation sequence and activates target gene. [2] Removing the inducer stops
transcription.[2]
Because a small inducer molecule is required, the increased expression of the target gene is
called induction.[2] The lactose operon is one example of an inducible system

49) Define Gene regulation(C212.5) (PO1,2,3,4)

Gene regulation is the informal term used to describe any

mechanism used by a cell to increase or decrease the production of
specific gene products (protein or RNA). Cells can modify their gene
expression patterns to trigger developmental pathways, respond to
environmental stimuli, or adapt to new food sources. All points of gene
expression can be regulated. This includes transcription, RNA
processing and transport, translation and post-translational modification
of a protein, and mRNA degradation.
50. Give the role of three enzyme of Lac-operon(C212.5) (PO1,2,3,4)
The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide,
into glucose and galactose. lacY encodes Beta-galactoside permease, a membrane
protein which becomes embedded in the cytoplasmic membraneto enable the cellular
transport of lactose into the cell. Finally, lacA encodes Galactoside acetyltransferase.
 PART B

S.N QUESTIONS REFER PAGE

O ENCE NO
UNIT I – CHEMISTRY OF NUCLEIC ACIDS
1 Explain the Structure and physicochemical properties of elements in DNA and TB1 80-82
RNA (May 2019)
2 Describe in detail the Biological significance of differences in DNA and RNA TB1 84-92
3 Write short notes on Primary structure of DNA. Describe the Chemical and TB1 94-96
structural qualities of 3’,5’-Phosphodiester bond
4 Describe in detail the Secondary Structure of DNA by Watson & Crick TB1 97-100
model
5 Explain the following: Triple helix, Quadruple helix, Reversible denaturation TB1 112-113
and hyperchromic effect
6 What is Tertiary structure of DNA? Explain DNA supercoiling. TB1 113-114
7 Give a detailed account on DNA as a genetic material, emphasizing their structure TB1 79
8 Explain structural feature is shared by both uracil and thymine? TB1 97-100

9 Explain GDP and AMP TB1 148

10 Explain CTP and dTDP TB1 148
UNIT II – DNA REPLICATION & REPAIR
1 Explain the experiment that proves semiconservative mode of replication (May TB1 225-228
2019)
2 Explain the role of Inhibitors in DNA replication (May 2019) TB1 232-235
3 Differentiate between prokaryotic and eukaryotic DNA replication TB1 271-273
4 How are okazaki fragments generated with diagrams? TB1 245, 248-
250
5 Demonstrate the rolling circle mode of replication (May 2019) TB1 255-258
6 What are the physical and chemical agents causing DNA mutations? TB1 300

7 Give any two repair mechanisms that rectify the error due to mutations TB1 306

8 Enlist the enzymes involved in prokaryotic DNA replication & TB1 209
comment
on their function.
9 Comment on initiation, elongation & termination events in TB1 271-273
prokaryotic
DNA replication.
10 Describe long patch repair mechanism TB1 293-305

UNIT III – TRANSCRIPTION

1 Elucidate the Structure and function of mRNA, rRNA and tRNA TB1 117
2 Narrate the Characteristics of promoter and enhancer sequences TB1 316
3 Explain the process of RNA synthesis and the Proteins involved in RNA TB1 317-329
synthesis
4 Explain the Differences in prokaryotic and eukaryotic transcription (May 2019) TB1 343-349
5 Explain RNA processing and 5’-Capping, TB1 352-358

6 Describe the process of Alternative splicing, Poly ‘A’ tail addition and base TB1 358-363
Modification (May 2019)
7 What is splicing? Explain Intron with self splicing activity. TB1 352-363

8 Give a detailed account on prokaryotic transcription initiation, elongation and TB1

termination mechanisms with suitable diagram and factors involed in it 317-329
9 What is transcription attenuation? Give an example of the operon regulated TB1 342
by this process.

10 Explain about the subunits are there in E.coli RNA polymerase . Add a note TB1 316
on core enzyme and holo enzyme of E.Coli RNA polymerase?

UNIT IV – TRANSLATION
1 How will you Elucidate genetic code? What is Codon degeneracy? TB1 367-378
2 Write notes on Prokaryotic and eukaryotic ribosomes TB1 439-447
3 Narrate the Steps in translation (May 2019) TB1 425-435
4 Explain the role of Inhibitors of protein synthesis. (May 2019) TB1 441-443
5 What are Post- translational modifications? (May 2019) TB1 443-445
6 Explain about origin and evolution of genetic code TB1 369
7 Describe nature & properties of genetic code. TB1 367-378
8 Explain the different properties of genetic code. TB1 367-378
9 What is Chromatin? Explain its organizatin. TB1 439-447
10 Explain the process of nucleosome formation. TB1 439-447
UNIT V – REGULATION OF GENE EXPRESSION
1 Explain the Organization of genes in prokaryotic and eukaryotic chromosomes TB1 453-454,
(May 2019) 502-510
2 Explain the Prokaryotic gene regulation in lac operon (May 2019) TB1 456-462
3 Explain the Prokaryotic gene regulation in trp operon TB1 479 – 480
4 Explain the Regulation of gene expression with reference to λ phage life cycle TB1 598 – 613
5 What is operon concept? Explain in detail ara Operon in Ecoli
6 Explain Hierarchical levels of gene regulation TB1 502-510
7 Comment on Regulation of operon TB1 456-462,
479 – 480
8 Explain Catabolite repression TB1 456-462,
479 – 480
9 Describe operator & structural gene TB1 456-462,
479 – 480
10 Distinguish between Positive & Negative Regulation TB1 456-462,
479 – 480

PART C
S.N QUESTIONS REFER PAGE
O ENCE NO
UNIT I – CHEMISTRY OF NUCLEIC ACIDS
1 Describe the secondary structure of DNA with diagrams TB1 97-100
2 Give an account on the forces that stabilises DNA TB1 110-112
3 Give direct evidence that DNA is genetic material TB1 79
4 List the biologically significant difference in DNA & RNA TB1 84-92
5 Discuss Watson and Crick model of DNA (May 2019) TB1 97-100
UNIT II – DNA REPLICATION & REPAIR

1 Explain the Organization of prokaryotic and eukaryotic chromosomes TB1 209

2 What are Mutagens? Describe in detail the various types of repair mechanisms TB1 293-305
(May 2019)
3 Explain Telomere replication in eukaryotes TB1 239-246
4 Distinguish between DNA polymerase I & DNA polymerase – II. TB1 209
5 What is physical mutagen? Explain on pyrimidine dimer formation TB1 293-305
UNIT III – TRANSCRIPTION
1 Describe the events of transcription with examples TB1 315

2 How do splicing mechanism favours processing of primary transcripts? TB1 359

3 Describe in detail about the post transcriptional modification in mRNA TB1 352-363

4 Distinguish between prokaryotic and eukaryotic transcription TB1 317-329

5 Outline the synthesis of mRNA with a neat sketch TB1 117

UNIT IV – TRANSLATION
1 Explain genetic code dictionary and the properties of it and mechanisms of TB1 369
regulation
2 Describe the events of prokaryotic translation with diagram TB1 430-435
3 What is the concept of genetic code? Describe the wobble hypothesis TB1 367
4 Explain the role of a) IF1 b) IF2 c) IF 3 d) EF Tu & EF Ts e) RF TB1 425-435
5 Explain activation of amino acids. TB1 367-378
UNIT V – REGULATION OF GENE EXPRESSION
1 Explain the organisation of eukaryotic chromosome with diagrams TB1 181
2 Lac operon is highly regulated. How? And give its implication in the generation TB1 460
of recombinant proteins
3 Explain eukaryotic gene regulation TB1 453-454,
502-510
4 Describe the detail mechanism of Lac operon. TB1 456-462,
479 – 480
5 Comment on impotrance of Catabolite activator protein in lac operon TB1 456-462,
functioning. 479 – 480