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  1. Transcription Dr.S.Chakravarty, MD

  2. Learning objectives • List the types of RNA, its structure and functions • Classify the types of RNA polymerases and its functions • Outline the steps of RNA transcription and its clinical applications • Processing of RNA in the nucleus and P-bodies • Differentiate between prokaryotic and eukaryotic ribosomes and its RNA • List the Drugs acting on transcription

  3. Transcription The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.

  4. Similarity between replication and transcription • Both processes use DNA as the template. • Phosphodiester bonds are formed in both cases. • Both synthesis directions are from 5´ to 3.

  5. Differences between replication and transcription

  6. Template and Enzymes

  7. The whole genome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the development requirement, physiological need and environmental changes. • DNA regions that can be transcribed into RNA are called structural genes.

  8. The coding strand is the strand whose base sequence specifies the amino acid CODING STRAND – SENSE STRAND TEMPLATE STRAND - ANTI SENSE STRAND

  9. coding strand template strand promoter promoter template strand coding strand

  10. Asymmetric transcription • Only the templatestrand is used for the transcription, but the coding strand is not. • Both strands can be used as the templates. • The transcription direction on different strands is opposite. • This feature is referred to as the asymmetric transcription.

  11. Organization of coding information in the adenovirus genome

  12. RNA Polymerase • The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase. • The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD. • Eukaryotic systems have three kinds of RNA polymerases, each of which is a multiple-subunit protein and responsible for transcription of different RNAs.

  13.      Holoenzyme The holoenzyme of RNA-pol in E.coli consists of 5 different subunits: 2.

  14. RNA-pol of E. Coli

  15. Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the  subunit of RNA-pol, and inhibit the RNA synthesis. • RNA-pol of other prokaryotic systems is similar to that of E. coli in structure and functions.

  16. The mushroom Amanita phalloides (The Death Cap Mushroom) produces a toxin called alpha-amanitin which is a potent inhibitor of RNA polymerase II. There are four clinical phases of poisoning: Asymptomatic phase Gastrointestinal phase (1-2 days) - severe diarrhea and vomiting Apparent recovery phase - few symptoms Hepatic phase - renal and liver failure *** death may occur in 7-10 days Treatment: -Gastrointestinal decontamination -high dose penicillin (inhibits amanitin in liver) -LIVER TRANSPLANT

  17. RNA-pol of eukaryotes USMLE FAVOURITE! Amanitin is a specific inhibitor of RNA-pol.

  18. Promoter The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control.

  19. Prokaryotic promoter Consensus sequence-A theoretical representative nucleotide or amino acid sequence in which each nucleotide is the one which occurs most frequently at that site in the different sequences which occur in nature

  20. Consensus Sequence

  21. Transcription Process

  22. The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process. • The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.

  23. Transcription of Prokaryotes • Initiation phase: RNA-pol recognizes the promoter and starts the transcription. • Elongation phase: the RNA strand is continuously growing. • Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

  24. a. Initiation • RNA-pol recognizesthe TTGACA region(-35), and slides to the TATAAT(-10) region, then opens the DNA duplex. • The unwound region is about 171 bp.

  25. The first nucleotide on RNA transcript is always purine triphosphate. ( GTP is more often than ATP. ) • The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed. • The three molecules form a transcription initiation complex. RNA-pol (2) - DNA - pppGpN- OH 3

  26. No primer is needed for RNA synthesis. • The  subunit falls off from the RNA-pol once the first 3,5 phosphodiester bond is formed. • The core enzyme moves along the DNA template to enter the elongation phase.

  27. b. Elongation • The release of the  subunit causes the conformational change of the core enzyme.The core enzyme slides on the DNA template toward the 3 end. • Free NTPs are added sequentiallyto the 3-OH of the nascent RNA strand.

  28. RNA-pol, DNA segment of ~40nt and the nascent RNA form a complex called the transcription bubble. • The 3segment of the nascent RNA hybridizes with the DNA template, and its 5end extends out the transcription bubble as the synthesis is processing.

  29. Transcription bubble

  30. RNA-pol of E. Coli

  31. Prokaryotic Transcription Unit

  32. Simultaneous transcriptions and translation

  33. Prokaryotic Polycistronic mRNA 3’ 5’ 5’ UTR Gene 1 Gene 2 Gene 3 3’ UTR 5’ 3’ Transcription AUG UAG AUG UAA AUG UGA Gene 1 Gene 2 Gene 3 5’ 3’ Translation Shine dalgarno protein 1 Protein 2 Gene 3 NH2 COOH NH2 COOH NH2 COOH

  34. c. Termination • The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex. • The termination occurs in either -dependent or -independent manner.

  35. The termination function of factor The factor, a hexamer, is a ATPase and a helicase.

  36. -independent termination • The termination signal is a stretch of 30-40 nucleotides on the RNA transcript, consisting of many GC followed by a series of U. • The sequence specificity of thisnascent RNA transcript will form particular stem-loop structures to terminate the transcription.

  37. Stem-loop disruption • The stem-loop structure alters the conformation of RNA-pol, leading to the pause of the RNA-pol moving. • Then the competition of the RNA-RNA hybrid and the DNA-DNA hybrid reduces the DNA-RNA hybridstability, and causes the transcription complex dissociated. • Among all the base pairings, the most unstable one is rU:dA.

  38. Transcription of Eukaryotes a. Initiation • Transcription initiation needs promoter and upstream regulatory regions. • The cis- acting elementsare the specific sequences on the DNA template that regulate the transcription of one or more genes.

  39. Transcription in Eukaryotes

  40. Cis-acting element

  41. Transcription factors • RNA-pol does not bind the promoter directly. • RNA-pol II associates with six transcription factors, TFII A - TFII H. • The trans-acting factors are the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.

  42. TF for eukaryotic transcription

  43. TAF TBP TF II H Pre-initiation complex (PIC) TBP of TFII D binds TATA TFII A and TFII B bind TFII D TFII F-RNA-pol complex binds TFII B TFII F and TFII E open the dsDNA (helicase and ATPase) TFII H: completion of PIC

  44. Phosphorylation of RNA-pol • TF II H is of protein kinase activity to phosphorylate CTD of RNA-pol. (CTD is the C-terminal domain of RNA-pol) • Only the p-RNA-pol can move toward the downstream, starting the elongation phase. • Most of the TFs fall off from PIC during the elongation phase.

  45. b. Elongation • The elongation is similar to that of prokaryotes. • However, transcription and translation do not take place simultaneously since they are separated by nuclear membrane.