1. LECTURE 14 RNA: TRANSCRIPTION & PROCESSING kchapter 8 k announcements k key ideas k RNA k transcription in Prokaryotes k transcription in Eukaryotes

2. ANNOUNCEMENTS g missed or late for quiz ? g same documentation required as for exams g no makeup… average of those you write g exam 2 … M 11.13 in class, as listed on calendar g extra tutorial help … g M 3:00 – 4:30 & F 11:00 – 12:30 in WHI 111 g extra-credit tutoring … in progress, check web page g extra-credit seminar WHI AUD F 3:30 g Dr. Juli Wade, Michigan State U. g Hormonal and Genetic Influences on Sexual Differentiation of the Zebra Finch Song System

3. CH8 KEY IDEAS k 3 processes of information transfer in genetics CH8  CH7  CH9 

4. CH8 KEY IDEAS k translation requires transfer RNAs & ribosomes k information transfer by non-overlapping triplet code k special DNA sequences signal initiation & termination of transcription & translation k in Eukaryotes only... k mRNA transcripts are processed prior to translation k noncoding introns interrupt coding exon sequences k introns are spliced out of 1° mRNA  final mRNA k DNA transcription RNA translation protein

5. CH8 KEY IDEAS k transcription & translation

6. RNA k DNA  protein... RNA intermediate? k pulse-chase experiments with RNA precursors... pulse radioactive, chase nonradioactive, autoradiograph k  synthesis of RNA in nucleus  protein in cytoplasm

7. RNA

11. k usually single-stranded,  double-stranded, complex 3D structures antiparallel k ribose sugar  deoxyribose C2 HC2 H C 2 OH RNA A U A T RNA DNA k uracil pyrimidine  thymine

12. k classes 1. informational k mRNA:translated  polypeptides 2. functional k tRNA: bind & transport amino acids k rRNA:components of ribosomes k snRNA:participate in modifying rRNA components of spliceosomes RNA

13. TRANSCRIPTION IN PROKARYOTES k T2 bacteriophage infection of E. coli k pulse-chase with radiolabeled uracil (RNA-precursor) k labeled RNA recovered only immediately after pulse...  rapid turnover k phage-induced RNA similar to T2 DNA...  DNA  RNA

14. TRANSCRIPTION IN PROKARYOTES k in vitro synthesis shows RNA  DNA...  DNA template & complementary RNA bases k similar to DNA replication, with DNA polymerase...  transcription enzyme... RNA polymerase ?

15. TRANSCRIPTION k RNA synthesized from 1 or both DNA strands? k hybridization experiment k complementary DNA strands have different densities k each RNA hybridizes to only 1 DNA strand k transcription is asymmetrical

16. TRANSCRIPTION IN PROKARYOTES k DNA template for mRNA transcription k note directional nature of event

17. TRANSCRIPTION IN PROKARYOTES k DNA coding strand = nontemplate strand k sequence homologous to transcribed mRNA k DNA has A, RNA has U k DNA template strand  mRNA

18. TRANSCRIPTION IN PROKARYOTES knew mRNA grows 5'  3' kRNA pol moves along DNA template strand 3'  5' 5' 3' 5' 3' error! k genes transcribed from one but either DNA strand k transcription by complementary pairing of bases k catalyzed by RNA polymerase (RNA pol)

19. TRANSCRIPTION IN PROKARYOTES kRNA pol moves along DNA template strand 3'  5' knew mRNA grows 5'  3' knormally illustrate in other direction for convenience

20. TRANSCRIPTION IN PROKARYOTES k transcription of rRNA genes in Triturus nucleus

21. TRANSCRIPTION IN PROKARYOTES k3 distinct stages of transcription 1. initiation 2. elongation 3. termination

22. TRANSCRIPTION IN PROKARYOTES kinitiation k2 types of sequences in prokaryotic genes 1. promoter sequences signal initiation 2. coding sequences are transcribed

23. TRANSCRIPTION IN PROKARYOTES kinitiation k2 regions of homology among promoter sequences kconsensus sequences = RNA pol binding sites

24. TRANSCRIPTION IN PROKARYOTES kinitiation k  factor binds to –10 and –35 consensus regions kinitiates melting or denaturing of DNA ktranscription begins when  subunit dissociates open promoter complex k different  factors recognize different DNA sequences closed promoter complex

25. TRANSCRIPTION IN PROKARYOTES kelongation knew mRNA grows 5'  3' kRNA pol and transcription bubble moves 3'  5' along the DNA template strand 5' 3'

26. TRANSCRIPTION kelongation knew mRNA grows 5'  3' kRNA pol moves along DNA template strand 3'  5' kribonucleoside triphosphate added to 3' end of n th base 3'3' 5'5' 5'5' 3'3'

27. kelongation knew mRNA grows 5'  3' kRNA pol moves along DNA template strand 3'  5' k pyrophosphate ion released 3'3' 3'3' 5'5' 5'5' TRANSCRIPTION IN PROKARYOTES

28. ktermination kRNA pol recognizes signal for chain termination k 2 mechanisms for termination in prokaryotes 1. direct termination - termination sequence TRANSCRIPTION IN PROKARYOTES mRNA 3' UTR DNA CG rich + AAA...(6+) 5' 3'

29. ktermination kdirect termination, RNA pol recognizes ~40 bp terminator sequence on template kRNA forms hairpin loop kpoly-A tail bonds weak ksignal to release RNA pol k ATP-independent TRANSCRIPTION IN PROKARYOTES

30. ktermination kRNA pol recognizes signal for chain termination k 2 mechanisms for termination in prokaryotes 1. direct termination - termination sequence 2. rho-dependent TRANSCRIPTION IN PROKARYOTES

31. ktermination krho-dependent termination kno U-residues formed kno hairpin loop krho binds to rut site on RNA TRANSCRIPTION IN PROKARYOTES

32. ktermination krho-dependent termination kno U-residues formed kno hairpin loop krho binds to rut site on RNA krho “pulls” RNA from RNA pol

33. TRANSCRIPTION IN PROKARYOTES ktermination krho-dependent termination kno U-residues formed kno hairpin loop krho binds to rut site on RNA krho “pulls” RNA from RNA pol

34. TRANSCRIPTION IN EUKARYOTES kdifferences because of complexity in Eukaryotes 1. RNA synthesis

35. TRANSCRIPTION IN EUKARYOTES kProkaryotes: k RNA pol  all types of RNA k polycistronic mRNA kEukaryotes: k RNA pol I  rRNA (except 5S rRNA) k RNA pol II  monocistronic mRNA, some snRNA k RNA pol III  tRNA, 5S rRNA, some snRNA

36. TRANSCRIPTION IN EUKARYOTES kProkaryotes: k RNA pol only kEukaryotes: k RNA pol II + general transcription factors (GTFs)  mRNA DNA GTFs

37. TRANSCRIPTION IN EUKARYOTES kdifferences because of complexity in Eukaryotes 1. RNA synthesis 2. RNA processing

38. TRANSCRIPTION IN EUKARYOTES kProkaryotes: k no processing kEukaryotes: k processed before being transported to the cytoplasm

39. TRANSCRIPTION IN EUKARYOTES kdifferences because of complexity in Eukaryotes 1. RNA synthesis 2. RNA processing 3. chromosome organization 4. split genes

40. TRANSCRIPTION IN EUKARYOTES kProkaryotes: k naked DNA (nearly) kEukaryotes: k chromatin – euchromatin & heterochromatin

41. TRANSCRIPTION IN EUKARYOTES k3 distinct stages of transcription 1. initiation 2. elongation 3. termination

42. TRANSCRIPTION IN EUKARYOTES kinitiation kGTFs  TATA sequence before RNA pol II binding k attract RNA pol II k positions complex kGTFs added after  preinitiation complex k transcription bubble

43. TRANSCRIPTION IN EUKARYOTES kelongation kGTFs added after  preinitiation complex k transcription bubble k RNA pol II carboxyl tail domain (CTD) phosphorylated...

44. TRANSCRIPTION IN EUKARYOTES k2 types of processing 1. cotranscriptional

45. TRANSCRIPTION IN EUKARYOTES kcotranscriptional processing – CTD dependend k guanyltransferase adds 7'-methylguanosine “cap” to 5' end of mRNA k protects single stranded RNA from degradation

46. TRANSCRIPTION IN EUKARYOTES kcotranscriptional processing – CTD dependend k splicing by spliceosomes (... stay tuned)

47. TRANSCRIPTION IN EUKARYOTES ktermination k AAUAAA sequence near 3' end initiates cleavage k... by endonuclease ~ 20 bp downstream

48. TRANSCRIPTION IN EUKARYOTES k2 types of processing 1. cotranscriptional 2. posttranscriptional

49. TRANSCRIPTION IN EUKARYOTES kposttranscriptional processing k poly(A) polymerase adds poly(A) tail of 150-200 adenosine residues to 3' end cleavage site k complete °1 mRNA

50. EUKARYOTIC RNA k°1 mRNA shortened before transport to cytoplasm kchicken ovalbumin DNA/mRNA hybrid  kcoding sequences... exons

51. EUKARYOTIC RNA k°1 mRNA shortened before transport to cytoplasm kchicken ovalbumin DNA/mRNA hybrid  kcoding sequences... exons kintervening sequences... introns

52. EUKARYOTIC RNA k°1 mRNA shortened before transport to cytoplasm kchicken ovalbumin DNA/mRNA hybrid  kcoding sequences... exons kintervening sequences... introns kintrons spliced from mRNA kgenomic DNA  RNA exons + introns

53. ksummary... kgenomic DNA  RNA exons + introns ktranscribed  1° mRNA transcript kprocessed  cap & polyadenylation kspliced  splicing intermediate kspliced  mature mRNA TRANSCRIPTION IN EUKARYOTES

54. k1 gene  multiple functions by alternative splicing kmultiple gene functions in different... k tissues k developmental stages kdifferent mRNAs from same 1° mRNA transcript... e.g.,  -tropomyocin gene... TRANSCRIPTION IN EUKARYOTES

55. k1 gene  multiple functions by alternative splicing e.g.,  -tropomyocin gene... TRANSCRIPTION IN EUKARYOTES

56. kgene splicing mechanism... ksequence homologies at exon-intron splice junctions k= consensus sequences for splicing enzymes... TRANSCRIPTION IN EUKARYOTES

57. kspliceosome ksnRNA aligns sequences correctly for splicing TRANSCRIPTION IN EUKARYOTES

58. ksplicing reaction kintron lariat structure kintrons excised by 2 transesterification reactions TRANSCRIPTION IN EUKARYOTES

59. ksplicing reaction kintron lariat structure kintrons excised by 2 transesterification reactions ksnRNA + proteins  small ribonuclear particles (snRNPs) ksnRNP + 1° mRNA transcript  spliceosome kspliceosome catalyzes splicing transesterification reactions TRANSCRIPTION IN EUKARYOTES

60. kself splicing mechanisms, mRNA catalyzes reaction (called ribozymes) TRANSCRIPTION IN EUKARYOTES

61. kself splicing mechanisms, mRNA catalyzes reaction kspliceosome-catalyzed mechanism TRANSCRIPTION IN EUKARYOTES

62. kTUTORIAL 6 QUESTIONS – CHAPTER 8