B.Tech Biotechnology II Elements of Biotechnology Unit 2 Structure of DNA

Elements of Biotechnology
Unit 2
B.Tech Biotechnology II
Basic concepts of
Genes, DNA & RNA
1

BREAKTHROUGH DISCOVERY
• In 1953, James Watson and Francis Crick
discovered the double helical structure of the
DNA molecule
2

DNA
A purine always links with a pyrimidine base to maintain the structure of DNA.
Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.
Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
3

Nucleoside & Nucleotide, the
fundamental building block of DNA
glycosidic bond
phosphoester bond
6

8
• Nucleotides have three characteristic
components:
• (1) a nitrogenous (nitrogen-containing) base,
(2) a pentose, and (3) a phosphate.
• The molecule without the phosphate group is
called a nucleoside.
• The nitrogenous bases are derivatives of two
parent compounds, pyrimidine and purine.

DNA & RNA - Nucleotide Bases
10

Nucleotide
adenine
deoxyribose
PO4
11

CHEMICAL AND PHYSICAL
PROPERTIES OF DNA
13

PHYSICAL PROPERTIES OF DNA
• DNA Stores Genetic Information
• Many lines of evidence show that DNA bears
genetic information. In particular, the Avery-
• MacLeod-McCarty experiment showed that
DNA isolated from one bacterial strain can
enter and transform the cells of another strain,
endowing it with some of the inheritable
characteristics of the donor. The Hershey-
Chase experiment showed that the DNA of a
bacterial virus, but not its protein coat, carries
the genetic message for replication of the virus
in a host cell. 14

DNA Is a Double Helix
• Putting together much published data, Watson
and Crick postulated that native DNA
consists of two antiparallel chains in a right-
handed double-helical arrangement.
Complementary base pairs, A=T and G C,
are formed by hydrogen bonding within the
helix. The base pairs are stacked perpendicular
to the long axis of the double helix, 3.4 Å apart,
with 10.5 base pairs per turn.
15

• DNA Sequences Adopt Unusual
Structures
• A number of other sequence-dependent
structural variations have been detected within
larger chromosomes that may affect the
function and metabolism of the DNA segments
in their immediate vicinity.
16

• A rather common type of DNA sequence is a
palindrome.
• A palindrome is a word, phrase, or sentence
that is spelled identically read either forward or
backward; two examples are ROTATOR
and NURSES RUN.
• The term is applied to regions of DNA with
inverted repeats of base sequence having
twofold symmetry over two strands of DNA
17

18
Such sequences are self-complementary within each strand and therefore
have the potential to form hairpin or cruciform (cross-shaped) structures.
When the inverted repeat occurs within each individual strand of the DNA,
the sequence is called a mirror repeat.
Mirror repeats do not have complementary sequences within the same strand
and cannot form hairpin or cruciform structures.

A, B and Z DNA
• A form – favored by
RNA
• B form – Standard
DNA double helix under
physiological conditions
• Z form – laboratory
anomaly,
– Left Handed
– Requires Alt. GC
– High Salt/ Charge
neutralization
19

• Four DNA strands can also pair to form a
tetraplex (quadruplex), but this occurs readily
only for DNA sequences with a very high
proportion of guanosine residues.
• The guanosine tetraplex, or G tetraplex, is
quite stable over a wide range of conditions.
• H-DNA, is found in polypyrimidine or
polypurine tracts that also incorporate a mirror
repeat. A simple example is a long stretch of
alternating T and C residues. The H-DNA
structure features the triple-stranded form
illustrated in Figure. Two of the three strands
in the H-DNA triple helix contain pyrimidines
and the third contains purines. 22

CHEMICAL PROPERTIES OF DNA
• ABSORPTION
• The bases in DNA absorb ultraviolet light at
the wavelength of 260 nm
• This absorption can be monitored using a
spectrophotometer
• This is one method used to figure the
concentration of DNA in solution
• The more DNA present, the higher the
absorption
23

• DENSITY
• Density can be measured by CsCl-density
ultracentrifugation
• can be used to estimate G+C content
• GC base pairs are more dense than AT base
pairs
• Density studies show the existence of satellite
DNA
24

• DENATURATION
• DNA is considered denatured when the double
stranded DNA molecule is converted into two
single stranded molecules
• As thermal energy increases, the frequency of
hydrogen bonds breaking between the molecules
increases
• G-C base pairs are held together by three
hydrogen bonds (A-Ts by two) and it therefore
takes more energy (higher temperatures) to
separate molecules with high GC contents
25

DNA can Form Hybrids
• The ability of two complementary DNA strands to
pair with one another can be used to detect similar
DNA sequences in two different species or within the
genome of a single species.
• Hybridization techniques can be varied to detect a
specific RNA rather than DNA. The isolation and
identification of specific genes and RNAs rely on
these hybridization techniques. Applications of this
technology make possible the identification of an
individual on the basis of a single hair left at the scene
of a crime or the prediction of the onset of a disease
decades before symptoms appear
26

27
DNA hybridization.
Two DNA samples to be compared are completely denatured by heating. When the two
solutions are mixed and slowly cooled, DNA strands of each sample associate with their
normal complementary partner and anneal to form duplexes. If the two DNAs have
significant sequence similarity, they also tend to form partial duplexes or hybrids with each
other: the greater the sequence similarity between the two DNAs, the greater the number of
hybrids formed. Hybrid formation can be measured in several ways.
One of the DNAs is usually labeled with a radioactive isotope to simplify the
measurements.

Nucleotides and Nucleic Acids Undergo
Nonenzymatic Transformations
• Purines and pyrimidines, along with the nucleotides of
which they are a part, undergo a number of
spontaneous alterations in their covalent structure.
• The rate of these reactions is generally very slow, but
they are physiologically significant because of the cell’s
very low tolerance for alterations in its genetic
information.
• Alterations in DNA structure that produce permanent
changes in the genetic information encoded therein are
called mutations, and much evidence suggests an
intimate link between the accumulation of mutations in
an individual organism and the processes of aging and
carcinogenesis.
28

• Hydrophobicity of solvent
• Hydrophobic substances will allow the bases in
DNA to dissolve into the solvent
• Whereas hydrophilic substances will keep the
bases of DNA stacked upon one another in
the orientation that most favors hydrogen
bonding between DNA strands
29

• pH
• Acidic pH cause breakage of phosphodiester
bonds between nucleotides and breakage of
the N-glycosidic bond between the sugar and
purine bases
• Alkali - Above pH 11.3, all hydrogen bonds
are disrupted and the DNA is totally
denatured
• Salts will stabilize the DNA double helix
30

• Electrophoresis
• DNA has a negative charge that is proportional to
its size
• This is due to the negatively charged phosphates
in the sugar-phosphate backbone
• If DNA is placed in an electrical field it will migrate
towards the positive electrode (the cathode)
• smaller pieces will migrate faster than larger pieces
• Larger pieces have trouble squeezing through the
gel matrix and are hence retarded while smaller
pieces migrate easier
31

• Type of gels
• Agarose is used to separate fairly large DNA
molecules
– 5 million to a few thousands base pairs
• Polyacrylamide is used to separate small pieces
of DNA
– 2 to several hundred base pairs
• The size of DNA is estimated by comparing
its migration through the gel to DNA
molecules of known size
32

RNA
Three major classes of RNA:
Difference between RNA & DNA
RNA DNA
RNA nucleotides contain ribose sugar DNA contains deoxyribose
RNA has the base uracil DNA has the base thymine
presence of a hydroxyl group at the 2'
position of the ribose sugar.
Lacks of a hydroxyl group at the 2'
position of the ribose sugar.
RNA is usually single-stranded DNA is usually double-stranded 33

mRNA
• Transcripts of structural genes.
• Encode all the information necessary for the
synthesis of a polypeptide of protein.
• Intermediate carrier of genetic information;
deliver genetic information to the cytoplasm.
34

mRNA to Amino Acid Dictionary
35

tRNA
 RNA molecules 70- 100 nucleotide long.
 The secondary structure of the tRNA
resembles a D loop, anticodon loop,
and T loop and the acceptor stem.
 Carry correct amino acids to their
position along the mRNA template to be
added to the growing polypeptide chain.
36

rRNA
• The central component of the
ribosome.
• Ribosome; factory for protein
synthesis; composed of ribosomal
RNA and ribosomal proteins.
• rRNA provides a mechanism for
decoding mRNA into amino acids.
• rRNA interact with the tRNAs during translation by
providing peptidyl transferase activity.
37

Biological roles of RNA
1. RNA is the genetic material of some viruses
2. RNA functions as the intermediate (mRNA)
between the gene and the protein-synthesizing
machinery.
3. RNA functions as an adaptor (tRNA) between
the codons in the mRNA and amino acids.
4. RNA serves as a regulatory molecule, which
through sequence complementarity binds to,
and interferes with the translation of certain
mRNAs.
5. Some RNAs are enzymes that catalyze essential
reactions in the cell (RNase P ribozyme, large
rRNA, self-splicing introns, etc).
38

Pseudoknots are complex structure
resulted from base pairing of
discontiguous RNA segments
Figure 6-32 Pseudoknot.
39

Structure (1): RNA chains fold back on
themselves to form local regions of double
helix similar to A-form DNA
RNASTRUCTURE(2)
hairpin
bulge
loop
RNA helix are the base-
paired segments between
short stretches of
complementary sequences,
which adopt one of the
various stem-loop
structures
40

Organization of DNA in eukaryotes
41

42
• Genome
Gene
• Is the basic units of
inheritance; it is a segment
within a very long strand of
DNA with specific
instruction for the
production of one specific
protein.
• Genes located on
chromosome on it's place or
locus.
Genome and Gene
• Totality of genetic information of an organism.
• Encoded in the DNA (for some viruses, RNA).

Chargaff’s rule of equivalance
A=T and G=C
43

Chargaff's rule
• Chargaff's rules state 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.
• They were discovered by Austrian
chemist Erwin Chargaff
44

• Deoxyribonucleic acid (DNA) is the
genetic material found in the
chromosomes of all animals and plants.
• It is made up of only four types of
organic nitrogenous bases: adenine (A),
guanine (G), thymine (T) and cytosine
(C).
• Of these, A and G are the purines and
T and C are the pyrimidines
45

• Chargaff gave the base pairing rule or the rule
of base equivalence which states that only one
purine can combine with one pyrimidine.
• That means A can combine with T and G with
C.
46

Experiment
• Chargaff and his students collected numerous DNA
samples for various organisms. Using the fairly new
technique of paper chromatography, Chargaff and his
associates proceeded to separate DNA.
• The DNA that they collected was subjected to acid.
The acid would then hydrolyze the phospodiester
bonds as it would cause a nucleophilic attack on the
bond and result in the backbone breaking up. Once the
phosphodiester bonds were broken then the individual
nucleotides would then be separated and be free to
analyze. Ultraviolet spectrophotometry was used to
analyze the exact amounts of bases that were present in
the DNA sample.
47

Relative proportions (%) of bases in DNA
Organism %A %G %C %T A/T G/C %GC %AT
φX174 24 23.3 21.5 31.2 0.77 1.08 44.8 55.2
Maize 26.8 22.8 23.2 27.2 0.99 0.98 46.1 54
Octopus 33.2 17.6 17.6 31.6 1.05 1 35.2 64.8
Chicken 28 22 21.6 28.4 0.99 1.02 43.7 56.4
Rat 28.6 21.4 20.5 28.4 1.01 1 42.9 57
Human 29.3 20.7 20 30 0.98 1.04 40.7 59.3
Grasshoppe
r 29.3 20.5 20.7 29.3 1 0.99 41.2 58.6
Sea Urchin 32.8 17.7 17.3 32.1 1.02 1.02 35 64.9
Wheat 27.3 22.7 22.8 27.1 1.01 1 45.5 54.4
Yeast 31.3 18.7 17.1 32.9 0.95 1.09 35.8 64.4
E. coli 24.7 26 25.7 23.6 1.05 1.01 51.7 48.3
48

Circular and super helical DNA
49

• Enzymes called topoisomerases can take apart
a circular DNA, introduce additional twists
into it, and then reseal the structure.
• Adding twists to circular DNA introduces
tension into the molecule.
• The extra tension in circular DNA (or in linear
DNA whose ends are anchored to prevent
tension from being released) usually causes the
molecule to writhe to alleviate the tension. Like
an overwound rubber band, the circular DNA
assumes a new shape, called a supercoil.
50

Relaxed and supercoiled DNA molecules
• Supercoiling can be positive (additional twists
added beyond the normal amount for linear DNA)
or negative (reduced numbers of twists compared to
linear DNA).
51

• The unstrained circle contains the same number of
twists as linear DNA. It is under no superhelical
tension.
• To make the strained circle, one twist was removed
(compared to linear DNA) and the resulting circular
DNA is strained because it has the same number of
base pairs (105), but fewer numbers of turns (twists).
Thus, the strained circle has a higher number of base
pairs per turn than the unstrained circle.
• To relieve the strain, the strained molecule can
introduce another superhelical turn within itself, called a
writhe.
• After the writhe, the number of twists (turns) is 10 again
so the number of base pairs per turn is 10.5 again, too.
52

• The linking number (L) is simply sum of the
number of twists (T) and writhes (W) of a
molecule:
• L = T + W
• Consequently, the change in the linking number
is also equal to the change in the twists and
writhes for a molecule:
• ΔL = ΔT + ΔW
• The superhelical density is defined as Δ L/L0,
where L0 is the linking number of the DNA in
its unstrained (relaxed state).
53

• Many naturally occurring DNA molecules have superhelical
densities of about -0.06. To get an idea of what this means,
consider a hypothetical DNA molecule of 10,000 bp, which is in
the "classical" B form, with 10.0 bp/turn. Then L0 is 10,000
bp/(10.0 bp/turn), or 1000 turns.
• Each DNA strand crosses the other 1000 times in the relaxed
circle. If the topoisomerase gyrase twisted the molecule to
a superhelical density of -0.06, then L = -0.06 L0, or L = -60.
This change could be accommodated, for example, by the helix axis
writhing about itself 60 times in a left-hand sense, which would
correspond to W = -60, T = 0; the molecule would have 60 left-
hand superhelical turns.
• Alternatively, the twist of the molecule could change so that it had
940 turns in 10,000 bp (T = 940) or 10,000/940 = 10.64
bp/turn. This would correspond to W = 0, T = -60. Although any
combination of T and W that sums to -60 could occur, real
molecules release strain mainly by writhing into superhelical turns,
because it is easier to bend long DNA than it is to untwist it.
54

THE TOPOLOGICAL PROPERTIES OF
DNA HELP US TO EXPLAIN
– DNA COMPACTING IN THE NUCLEUS
– UNWINDING OF DNA AT THE
REPLICATION FORK
– FORMATION AND MAINTENANCE OF THE
TRANSCRIPTION
55

References
• Images references:
1-2 Lehninger Principles of biochemistry by Nelson and Cox
• Reading references:
• Gene cloning and DNA analysis by TA Brown
1

B.Tech Biotechnology II Elements of Biotechnology Unit 2 Structure of DNA

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