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Similar to Transcription
Transcription
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- 2. Definition Synthesis of RNAusing ssDNA as a template by DNA dependent RNA polymerase enzyme. Similar to replication in terms of chemical mechanism, polarity, and use of template but differs in -does not require primers -only a short segment of DNA is transcribed DNA RNA Protein TRANSCRIPTION TRANSLATION Nucleus
- 3. All 3 typesof cellular RNA’s are copied during transcription Messenger RNAs (mRNAs) encode the amino acid sequence of one or more polypeptides specified by a gene. Transfer RNAs (tRNAs) read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis. Ribosomal RNAs (rRNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins.
- 4. Salient features oftranscription Synthesis of ALL types of RNA in Nucleus Only ONE STRAND of DNA participates Ribonucleotides are used in RNA synthesis. RNA Synthesis occurs in 5’-3’ direction , DNA template is read from 3’-5’ direction Synthesis follows Waston-Crick base pairing rules – A to U, G to C DNA dependent RNA polymerase or RNA polymerase
- 5. • ss DNA Template •DNAdependent RNA polymerase Enzyme • ATP, GTP,CTP,UTPRibonucleoside triphosphates Basic Requirements of Transcription
- 6. Steps involved inTranscription INITIATION ELONGATION TERMINATION & Release
- 7. 1.Promoter region: Itis the specific region in DNA ,where transcription is initiated. 2.Transcription unit: It is the region where DNA template is transcribed. Present in b/w promoter and terminating units. 3.Termination unit: It is the region where transcription terminates. Template DNA DNA Promoter region Transcription unit Termination unit
- 8. Initiation Starts with therecognition of promoter sequence on the DNA coding ( anti- template ) strand by RNA polymerase Promoter sequence Are specific areas on the DNA that act as starting signals for initiation process recognized by RNAP. Two common sequences are present on the upstream side of the start site of transcription . Start site is denoted by +1
- 9. Pribnow box orTATAAT box: It contains 6 nucleotide bases TATAAT located -10 bases away on the left of origin of transcription. -35 sequence / 5`-TTGACA-3`seqence: it is 35 bp upstream of the transcription unit Promoters of prokaryotes TTGACA5` 3`
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- 11. RNAP- prokaryotes RNAP ofprokaryotes has 5 sub units-2 alpha units , beta, beta1 and sigma unit (2α,β,β’,δ). alpha Alpha` betaBeta` Sigma factor Core enzyme Beta Beta1 Alpha Alpha RNA polymerase without σ subunit is called core enzyme Core enzyme contains the catalytic activity. Core enzyme with sigma factor is holo-enzyme
- 12. Subunit Role α Bindsregulatory proteins β Forms phosphodiester bond β’ Fixes RNAP to DNA template σ Recognizes and binds to promoter region of DNA, Initiates transcription. Functions of RNAP In bacteria, one species of RNAP can synthesize all the RNA molecules ( mRNA, tRNA, rRNA )
- 13. RNA polymerase differsfrom DNA Polymerase in two aspects 1. No primer is required for RNAP. 2. RNAP lacks a separate proofreading 3’ to 5’ exonuclease activity. The error rate for transcription is higher. Error rate is 104-105 times more than replication Errors cause little damage – as they are not transmitted to daughter cell/ next generation.
- 14. Identification of promoterregion: Sigma unit of RNA Polymerase identifies the promoter region on template DNA . RNA polymerase binds to the promoter region of the template DNA Beta unit fixes to the promoter region of the template strand and initiates transcription.
- 15. Transcription bubble RNA Sigma factoris released and RNAP in promoter region unwinds DNA helix. A Transcription bubble is formed as the DNA unwinds down stream Transcription bubble contains RNAP, DNA and the new RNA formed Supercoils is overcome by Topoisomerase
- 16. Elongation RNA polymerase RNA nucleotide Direction of transcription Newlymade RNA Template strand of DNA RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’- hydroxyl end. RNAP uses ribonucleoside triphosphates, forms 3’5’ PDE bond b/w adjacent ribonucleotide and releases ppi each time a nucleotide is added to the growing chain Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interaction. (G to C, A to U)
- 17. Rho dependent termination- A hexameric protein called Rho factor attaches to the DNA strand and RNAP can not move further and dissociates from DNA strand terminating the transcription Rho independent termination – palindrome like bases occurs at end sequence of DNA. Due to these sequences, the newly synthesized RNA folds on itself to form hair pin loop (due to complemenatry basepairing) which terminates the movement of RNAP. palindrome – word / phrases that read alike backwards and forwards – MADAM, MALAYALAM Termination
- 18. Termination Rho- factor ATP ADP+ pi Rho factor is an ATP dependent RNA-DNA helicases Recognizes and bind to the termination signals and disrupts the nascent RNA/DNA complex
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- 22. Prokaryotes Eukaroytes Simple Morecomplex One RNAP 3 distinct RNAP Promoter site – Pribnow box 35 sequence Promoter site – TATA box – Hogness box , CAAT box Initiation – Only requires sigma factor Initiation – 6 Transcription factors interact with eukaryotic promoter region. POST TRANSCRIPTIONAL MODIFICATION
- 23. Promoters of eukaryotes Goldberg–hogness box; In eukaryotes a sequence TATAAA is located at 25-30 bp upstream to the start point it acts as signal to initiate the transcription. CAAT box : GGCAATCT Sequence is located 70 bp upstream to start point. TATAAA
- 25. RNAP of eukaryotesare of 3 types RNAP-I synthesizes - rRNA RNAP-II synthesizes - mRNA RNAP-III synthesizes - tRNA
- 26. Transcription factors bindsto DNA sequences in promoter region Stimulated by enhancers 6 Transcription factors – TFIID, TFIIA,TFIIB, TFIIF, TFIIE, TFIIH. Transcription factors binds to each other and in turn to enzyme RNAP – form preinitiation complex or basal transcription complex
- 27. Enhancer –increases gene expression by 100 fold Enhancers bind to TFs to form Activators They have no Promoter activity of their own but stimulate the transcription gene. They can be present upstream, downstream or within a gene Silencers – DNA sequences which bind proteins that act to inhibit the rate of transcription.
- 31. Post transcriptional modifications ThemRNA formed from DNA is called the primary transcript or hnRNA. It undergoes extensive modifications to become active and mature mRNA. These modifications are called as post transcriptional modifications. Eukaryotic RNA is processed before leaving the nucleus
- 32. hnRNA to mRNA 1.cappingat 5`end 2. Poly A tailing at 3’ 3.Splicing - removal of introns 4. mRNA EDITING
- 33. Post transcriptional modifications 5’capping 7-methylguanylate attached by a unusual 5’-5’ triphosphate linkage to the ribose at the 5’-end. Addition of GTP part of the cap is catalyzed by nuclear enzyme guanylyltransferase. Methylation of terminal guanine occurs in the cytosol- SAM is the source of the methyl group Catalysed by guanine-7-methyl transferase.
- 34. Importance The cap bindsmature mRNA to the ribosome during protein biosynthesis. Cap Stabilizes mRNAs against digestion by ribonucleases. Eukaryotic mRNAs lacking the cap are not translated efficiently.
- 35. Addition of polyA tail Poly A tail added at 3’ end of mRNA 200-300 adenylate residues linked by PDE bonds ATP is the donor of adenylate group It is involved in stabilization of mRNA
- 36. Splicing Process by whichintrons are removed & exons are joined to form the functional mRNA . Requires energy Small nuclear RNAs associated with specific proteins to form complex –snRNPs (small nuclear ribonucleic protein particles) or snurps , involved in formation of spliceosomes. Spliceosomes –is a complex containing multiple snRNPs that contain snRNA that catalyze hnRNA to mRNA, by removing introns and joining exons. 15% genetic disease – due to splicing defects Faulty splicing – causes βThalassaemia.
- 37. DNA RNA transcript with cap and tail mRNA ExonIntron IntronExon Exon Transcription Addition of cap and tail Introns removed Exons spliced together Coding sequence NUCLEUS CYTOPLASM Tail Cap
- 39. mRNA editing 0.01% ofthe mRNAs undergoes editing. enzyme mediated alteration of base sequence of RNA (not by splicing ) Ex:- conversion of CAA codon in mRNA (of apoprotein B gene) to UAA by the enzyme cytidine deaminase . Originating from the same gene, the liver synthesizes a 100-kDa protein (apoB 100) while the intestinal cells synthesize 48-kDa protein (apoB 48). This happens due to formation of a termination codon UAA from CAA in RNA editing.
- 40. tRNA Cleavage of a5’leader sequence. Splicing to remove intron. Replacement of the 3’terminal UU by CCA & Modification of several bases – dihydrouridine, pseudouridine, Thymine, methylated bases.
- 41. 28 s,18s, 5.8 s are synthesized as long precursor – Preribosomal 45S RNAs This is cleaved and trimmed to produce mature functional rRNA 5 S rRNA is produced by transcription of 5S gene by RNA polymerase III & modified separately. rRNA
- 42. Inhibitors Mode ofaction Rifampicin antitubercular drug Only inhibits prokaryotes binds to beta subunit of RNA polymerase Actinomycin D synthesized by Streptomyces Both Binds with DNA strand & blocks movements of all forms of RNA. a-Amanitin toxin produced by Mushroom, Amanita phalloides Eukaryotes tightly binds with RNA polymerase ll & inhibits transcription Inhibitors of transcription
- 43. Reverse transcription Generation ofDNA from RNA is reverse transcription Enzyme catalyzing this is reverse transcriptase or RNA dependent DNA polymerase Usually seen in RNA viruses of retrovirus group Some of the tumor viruses Eg: HIV causing AIDS is a retrovirus. genetic information is transferred from RNA to DNA
- 44. Ribozyme Enzymes made upof RNA are called ribozymes Catalytic RNA molecules with sequence specific cleavage activity exhibit Michaelis-Menten kinetics Ex:- Spliceosomes - contains ribozymes as well as protein. Involved in Post transcriptional modification. RNAse-P- generates the ends of tRNAs. Peptidyl transferase -used for protein biosynthesis