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2. Transcription Dr. Ishtiaq Ahmad Khan Dr. Panjwani Center for Molecular Medicine and Drug Research

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

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

5. replicationtranscription templatedouble strandssingle strand substratedNTPNTP primer yesno EnzymeDNA polymeraseRNA polymerase productdsDNAssRNA base pairA-T, G-CA-U, T-A, G-C 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. § 1.1 Template The template strand is the strand from which the RNA is actually transcribed. It is also termed as antisense strand. The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein. Therefore, it is also called as sense strand.

10. Only the template strand 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. Asymmetric transcription

12. § 1.2 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. subunitMWfunction  36512 Determine the DNA to be transcribed  150618Catalyze polymerization  155613Bind & open DNA template  70263 Recognize the promoter for synthesis initiation RNA-pol of E. Coli

15. RNA-polIIIIII products45S rRNAmRNA 5S rRNA tRNA snRNA Sensitivity to Amanitin Nohighmoderate RNA-pol of eukaryotes Amanitin is a specific inhibitor of RNA-pol.

16. Each transcriptable region is called operon. One operon includes several structural genes and upstream regulatory sequences (or regulatory regions). The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control. § 1.3 Recognition of Origins

17. Promoter

18. Prokaryotic promoter Consensus sequence

19. Consensus Sequence

20. The -35 region of TTGACA sequence is the recognition site and the binding site of RNA-pol. The -10 region of TATAAT is the region at which a stable complex of DNA and RNA-pol is formed.

21. Section 2 Transcription Process

22. General concepts Three phases: initiation, elongation, and termination. 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. § 2.1 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 recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex. The unwound region is about 17  1 bp.

25. 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.

26. 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 sequentially to the 3 -OH of the nascent RNA strand.

27. Transcription bubble

28. RNA-pol of E. Coli

32. Simultaneous transcriptions and translation

33. 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.

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

35.  -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 this nascent RNA transcript will form particular stem-loop structures to terminate the transcription.

36. § 2.2 Transcription of Eukaryotes Transcription initiation needs promoter and upstream regulatory regions. The cis-acting elements are the specific sequences on the DNA template that regulate the transcription of one or more genes. a. Initiation

37. Cis-acting element

38. TATA box

39. 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. Transcription factors

40. TF for eukaryotic transcription

41. 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 Pre-initiation complex (PIC)

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

43. The termination sequence is AATAAA followed by GT repeats. The termination is closely related to the post-transcriptional modification. c. Termination

44. Splicing

45. mRNA Splicing

49. Daniel A. Pomeranz Krummel, Chris Oubridge, Adelaine K. W. Leung, Jade Li & Kiyoshi Nagai Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution Nature 458, 475-480(26 March 2009) doi:10.1038/nature07851

50. Lehninger, Principles of Biochemistry, Fifth Edition - WH Freeman

51. Alternative RNA splicing Shortly after the discovery of splicing came the realization that the exons in some genes were not utilized in the same way in every cell or stage of development. In other words exons could be skipped or added. This means that variations of a protein (called isoforms) can be produced from the same gene.

52. Eukaryotic mRNA can be processed in more than one way to produce different mRNAs and thus different polypeptides. The primary transcript contains molecular signals for all the alternative processing pathways, and the pathway favored in a given cell is determined by processing factors, RNA binding proteins that promote one particular path.

53. Alternative splicing largely explains how the 20,000–30,000 genes in the human genome can Encode the hundreds of thousands of different proteins estimated to exist in human cells.