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Transcription and the various stages of transcription
- 1. Moderator : Dr Smita S Sonoli Speaker: Mohit Adhikary
- 2. Outline • Introduction • Prokaryotic transcription • Eukaryotic transcription • Post transcriptional modification
- 4. Difference between replication &transcription REPLICATION TRANSCRIPTION • New DNA is formed • DNA-DNA hybrid complex • DNA polymerase enz. • Primer is requried • Deoxyrinonucleotides used • Entire genome is copied. • Proofreading • Genetic information is inherited. • New RNA is formed • DNA –RNA hybrid complex • RNA polymerase enz. • Primer not requrired • Ribonucleotides used • Very small portion of genome transcribed • No proofreading. • Information is transferred from gene to protein.
- 5. 4 types of RNA • mRNA • rRNA • tRNA • smallRNA – snRNA – scRNA
- 6. Central dogma of molecular biology
- 8. Replication, transcription and translation
- 9. • The process in a cell by which genetic material is copied from a strand of DNA to a complementary strand of RNA. OR The synthesis of RNA from a DNA template. • TWO aspects of transcription must be considered • The enzymology • The signal that determine where on a DNA molecule transcription begins and stops. TRANSCRIPTION
- 11. Transcription
- 13. Multiple functions of RNA polymerase • Searches promoter sites • Unwinds DNA helical • Selection of correct ribonucleoside triphosphate • Detects termination signals • Interacts activator & repressor proteins • Essential function of RNAP in all cells make them attractive targets for antibiotics & other drugs.
- 14. Types of RNAP • Type I or A: – rRNA – Not inhibited by amantin • Type II or B: – main enzyme synthesizing mRNA – Inhibited by amanitin – snRNA & miRNA – Has 2 large and 12 smaller sub units – Activated by phosphorylation • Type III or C: – tRNA – Moderately sensitive to amanitin
- 15. Difference between DNA & RNA Polymerase • RNA P • No primer required • Does not posses endo & exonuclease activity • No proof reading • DNA P • Primer required • Posses endo & exonuclease activity • Proof reading done
- 16. Prerequisite of transcription Prokaryote • Substrate – NTPs • ATP • GTP • CTP • UTP • DNA dependent RNA Polymerase (2α 1β1β’ 1δ) with 2 Zn molecule Eukaryote • Substrate – NTPs • ATP • GTP • CTP • UTP • RNAP type I / A – rRNA (large) RNAP type II / B – mRNA & snRNA RNAP type III / C – tRNA & small rRNA
- 19. -35 5’ TTGACA-3’ -10 5’ TATAAT-3’ Pribnow box +1 Initiation Cyclic AMP receptor protein (CRP)
- 23. 3’ end 3’ end
- 24. 3’ end 5’ end PPi PPi PPi PPi PPi PPi PPi PPi PPi PPi PPi PPi PPiNTPs pause
- 26. Termination • Two types – Rho dependent termination – Rho independent termination
- 27. 3’ end 5’ end 5’ end Rho dependent termination
- 28. Rho independent termination • Formation of hair pins of newly synthesised RNA. • RNA self complementary
- 29. “N” represents a non-complementary base.
- 30. Action of antibiotics • Rifampicin • Dactinomycin
- 31. Transcription in Eukaryotes • Separate polymerases for synthesis of rRNA, tRNA, mRNA • Transcription factors
- 32. Chromatin structure & Gene expression • Association of DNA with histones • Acetylation of lysine residues at amino terminus of histone protein • Histone acetyltransferase • Histone deacetylase
- 33. Nuclear RNA polymerase of eukaryotic cells • RNA Polymerase I – Synthesizes the precursor of 28S, 18S & 5.8S rRNA • RNA Polymerase II • RNA Polymerase III • Synthesizes tRNA, 5SrRNA, snRNA & snoRNA • Mitochondrial RNA polymerase
- 34. RNA Polymerase II Promoters and transcriptional factors for RNA synthesis •TATA or Hogness box •CAAT box GC rich box •TFs •TFIID: recognises and binds the TATA box •TFFIF: brings polymerase to the promoter •TFIIH: •Helicase: melts DNA •Kinase: phosphorylates polymerase
- 36. Role of enhancer in gene regulation • Cis acting DNA sequence: inc. rate • +nt on the same chromosome • They can – Be located upstream or down stream of transcription start site – Be close or thousands of base pair away from the promoter – Occur on either strand of DNA
- 39. DNA Primary transcript (inactive) Functionally active RNA molecules transcription Alteration (splicing, base modification)
- 40. Ribosomal RNA • Synthesised by long precursor pre-ribosomal RNA • Prokaryotes 23s,16s,5s • Eukaryotes 28s,18s,5.8s •Later trimmed by exonucleases •Modified at some bases & ribose
- 41. Transfer RNA • An intron removed from anticodon loop by nucleases • Seq. of 3’ 5’ must be trimmed • Addition of CCA by nucleotidyltransferase • Modification of bases at specific position (dihydrouracil)
- 42. Before After
- 43. Messenger RNA Primary transcript subjected to: • 5’ capping • Poly A tail • Removal of introns • splicing
- 45. Poly A tail
- 46. Introns Exons
- 49. Systemic lupus erythematosus, an often fatal inflammatory disease, results from an autoimmune response in which the patient produces antibodies against host proteins, including snRNP.
- 50. Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that fifteen percent of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia—a disease in which the production of the β-globin protein is defective
- 51. Alternative splicing patterns in eukaryotic mRNA.
- 53. Reverse transcription RNA dependent DNA polymerase. retro viruses carry RNA as their genetic material & can synthesize double stranded DNA from their genomic RNA by process know Reverse transcription. Viral DNAP is called reverse transcriptase . Exa. HIV • Viral DNAP is called reverse transcriptase. • RNA dependent DNA polymerase. • Retroviruses carry RNA as their genetic material & can synthesize double stranded DNA from their genomic RNA by process REVERSE TRANSCRIPTION. • Eg. HIV
- 54. “We don’t see things as they are, we see things as we are.” -Anais Nin