1. Transcription and RNA processing Fall, 2015

2. Transcription Outline Notes RNA Polymerase Structures Subunits Template versus coding strands Polymerase movement. Promoters Prokaryotic sequences / -35 / -10 sequences Alternative promoters - governed by sigma factors Prokaryotic RNA Synthesis Transcription bubble

3. Termination signal ◦Stem loop ◦FMN-specific termination ◦Rho protein. Antibiotic inhibition ◦Rifampicin / Site of action - elongation blocker ◦Actinomycin D (binds DNA double helix - ) tRNA and rRNA Processing in prokaryotes ◦Ribonuclease P (generates 5' terminus of tRNAs) ◦Ribonuclease III (excises 5S, 16S, 23S rRNAs from primary transcript) ◦CCA addition to tRNAs ◦Base modifications Uridylate modifications

4. Eukaryotic RNA Synthesis Transcription/Translation - Prokaryotes vs. Eukaryotes RNA Polymerases Amanitin structure and source Promoter elements Eukaryotic TATA box / CAAT and GC boxes Transcription Initiation TATA-binding protein - DNA complex Transcription factor HSTF ◦Sequence recognized = 5' CNNGAANNTCCNNG 3' ◦Binding sites II

5. Enhancer sequences ◦No promoter activity of own ◦Act up to several thousand bp away from gene ◦Act upstream, downstream, in middle of gene, and orientation independent ◦Specific to specific cells Eukaryotic rRNA ◦Made by RNA Polymerase I as pre-rRNA ( and ) Eukaryotic tRNA ◦Made by RNA Polymerase III ◦Processing Eukaryotic mRNA ◦Made by RNA Polymerase

6. ◦Capping 5' end of mRNAs Polyadenylation 3' end of mRNAs microRNAs ◦made by RNA Pol II or III ◦processed from larger precursors ◦roles in controlling gene expression RNA editing ◦apo B-100/apo B-48 ◦Cation channel proteins ◦Trypanosomes (insertion of uridines after transcription using guide RNAs)

7. Splicing ◦Sites ( ◦Splicing mechanism ◦Lariat branch point ◦Spliceosome assembly ◦Splicing catalytic center ◦snRNPs ◦Transcription and processing coupled ◦Splicing mutations and disease Thalassemia Examples ◦Alternative splicing patterns (calcitonin/CGRP - ) ◦Self splicing intron Schematic ◦Splicing pathway comparisons

8. Definition of: transcription, replication, and translaytion

9. RNA POLYMERASE

10. DNA POLYMERASE AND RNA POLYMERASE Differences and similarities Template Primers Starting point Directions Proof reading

11. SUBUNITS

12. TEMPLATE VERSUS CODING STRANDS

13. PROMOTERS

14. -35/-10 SEQUENCES

15. Alternative promoters - governed by sigma factors

16. Prokaryotic RNA Synthesis Transcription bubble

17. phases Initiation: the first 10 nt, open complex, sigma leaves. Rifampicin bind RNA pol and prevent transfer from initiation to elongation Elongation: RNA pol fits and starts Termination

18. Antibiotic inhibition ◦Rifampicin

19. Termination signal ◦Stem loop

20. ◦FMN-specific termination

21. ◦Rho protein

22. tRNA and rRNA Processing in prokaryotes

23. Processing One long RNA transcript, and then the individual pieces are cleaved Ribonuclease P: chewing away 5 ‘ end and to make 5’ end of tRNA Ribonuclease III: cuts the 5s, 16s, and the 23s RNA CCA sequence is added to the 3’ end of tRNA

24. ◦Actinomycin D (binds DNA double helix

25. ◦Base modifications Uridylate modifications

26. tRNA is largly modified rRNA little modified mRNA very very little modified

27. Eukaryotic RNA Synthesis

28. Transcription/Translation - Prokaryotes vs. Eukaryotes

29. RNA Polymerases

30. Amanitin structure and source

31. Promoter elements

32. Eukaryotic TATA box :any between -35 to -100, not always t but genes which are made largely they will have it

33. CAAT and GC boxes

34. Enhancer sequences – No promoter activity of own – Act up to several thousand bp away from gene – Act upstream, downstream, in middle of gene, and orientation independent – Specific to specific cells

35. Transcription Initiation

36. TATA-binding protein - DNA complex

37. Transcription factor HSTF ◦Sequence recognized = 5' CNNGAANNTCCNNG 3' ◦Binding sites

38. Eukaryotic rRNA ◦Made by RNA Polymerase I as pre-rRNA

39. Eukaryotic tRNA ◦Made by RNA Polymerase III ◦Processing

40. Eukaryotic mRNA ◦Made by RNA Polymerase II ◦Capping 5' end of mRNAs : protective against nucleases, a handle for translation, and clear 5’ end

41. Polyadenylation 3' end of mRNAs

42. microRNAs ◦made by RNA Pol II or III ◦processed from larger precursors ◦roles in controlling gene expression

43. RNA editing ◦apo B-100/apo B-48 ◦Cation channel proteins ◦Trypanosomes (insertion of uridines after transcription using guide RNAs)

45. Splicing

46. ◦Sites

47. ◦Splicing mechanism

48. ◦Alternative splicing patterns (calcitonin/CGRP

49. ◦Spliceosome assembly

50. ◦Splicing catalytic center

51. ◦snRNPs

52. ◦Splicing mutations and disease Thalassemia

53. Examples

54. ◦Transcription and processing coupled