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LECTURE 14 RNA: TRANSCRIPTION & PROCESSING

LECTURE 14 RNA: TRANSCRIPTION & PROCESSING. chapter 8 announcements key ideas RNA transcription in Prokaryotes transcription in Eukaryotes. ANNOUNCEMENTS. missed or late for quiz ? same documentation required as for exams no makeup… average of those you write

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LECTURE 14 RNA: TRANSCRIPTION & PROCESSING

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

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

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

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

  5. CH8 KEY IDEAS • transcription & translation

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

  7. RNA

  8. RNA

  9. RNA

  10. RNA

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

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

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

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

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

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

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

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

  19. TRANSCRIPTION IN PROKARYOTES • normally illustrate in other direction for convenience • new mRNA grows 5'  3' • RNA pol moves along DNA template strand 3'  5'

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

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

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

  23. TRANSCRIPTION IN PROKARYOTES • initiation • 2 regions of homology among promoter sequences • consensus sequences = RNA pol binding sites

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

  25. TRANSCRIPTION IN PROKARYOTES • elongation • new mRNA grows 5'  3' • RNA pol and transcription bubble moves 3'  5'along the DNA template strand 5' 3'

  26. 3' 5' 3' 5' TRANSCRIPTION • elongation • new mRNAgrows 5'  3' • RNA pol moves along DNA template strand 3'  5' • ribonucleoside triphosphate added to 3'end of nth base

  27. 3' 5' 3' 5' TRANSCRIPTION IN PROKARYOTES • elongation • new mRNAgrows 5'  3' • RNA pol moves along DNA template strand 3'  5' • pyrophosphate ion released

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

  29. TRANSCRIPTION IN PROKARYOTES • termination • directtermination, RNA pol recognizes ~40 bp terminator sequence on template • RNA forms hairpin loop • poly-A tail bonds weak • signal to release RNA pol • ATP-independent

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

  31. TRANSCRIPTION IN PROKARYOTES • termination • rho-dependenttermination • no U-residues formed • no hairpin loop • rho binds to rut site on RNA

  32. TRANSCRIPTION IN PROKARYOTES • termination • rho-dependenttermination • no U-residues formed • no hairpin loop • rho binds to rut site on RNA • rho “pulls” RNA from RNA pol

  33. TRANSCRIPTION IN PROKARYOTES • termination • rho-dependenttermination • no U-residues formed • no hairpin loop • rho binds to rut site on RNA • rho “pulls” RNA from RNA pol

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

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

  36. TRANSCRIPTION IN EUKARYOTES • Prokaryotes: • RNA pol only • Eukaryotes: • RNA pol II + general transcription factors (GTFs)  mRNA DNA GTFs

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

  38. TRANSCRIPTION IN EUKARYOTES • Prokaryotes: • no processing • Eukaryotes: • processed before being transported to the cytoplasm

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

  40. TRANSCRIPTION IN EUKARYOTES • Prokaryotes: • naked DNA (nearly) • Eukaryotes: • chromatin – euchromatin & heterochromatin

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

  42. TRANSCRIPTION IN EUKARYOTES • initiation • GTFs  TATA sequence before RNA pol II binding • attract RNA pol II • positions complex • GTFs added after  preinitiation complex • transcription bubble

  43. TRANSCRIPTION IN EUKARYOTES • elongation • GTFs added after  preinitiation complex • transcription bubble • RNA pol II carboxyl tail domain (CTD) phosphorylated...

  44. TRANSCRIPTION IN EUKARYOTES • 2 types of processing • cotranscriptional

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

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

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

  48. TRANSCRIPTION IN EUKARYOTES • 2 types of processing • cotranscriptional • posttranscriptional

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

  50. EUKARYOTIC RNA • °1 mRNA shortened before transport to cytoplasm • chicken ovalbumin DNA/mRNA hybrid  • coding sequences... exons

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