Transcription

1. Transcription Chapter 26

2. Genes • Nucleotide seq’s w/in DNA • ~2000 genes for peptides in prokaryotes • ~50,000 genes for peptides in eukaryotes • DNA is not DIRECT template for peptides • DNA = template for RNA (specifically mRNA) • Synth mRNA from DNA = transcription • So DNA transcribed to mRNA • mRNA used to translate genetic code  peptide (next lecture)

3. RNA Is Similar to DNA • Both nucleic acids • Both composed of 4 nucleotides: A, G, C • BUT RNA has U, not T • Both have form of ribose sugar • BUT RNA has ribose, DNA deoxyribose • Both linked by phosphodiester bonds •  Sugar-phosphate backbone • BUT RNA in single-strand (not dbl helix) • Strand can fold back on itself • Can form intrastrand helices, other 2o structures

4. Transcription DNA  RNA Similar to Repl’n DNADNA • Complementarity • Base seq daughter DNA complementary to DNA template (parent) strand • Base seq mRNA complementary to DNA template strand • Initiation, Elongation, Termination Processes • Polymerases catalyze syntheses of new nucleic acid • Free 3’ –OH attacks –PO4 of incoming triphosphate • Pyrophosphate (PPi) is released • (NMP)n + NTP  (NMP)n+1 + PPi

5. Transcription DNA  RNA Similar to Repl’n DNADNA • Template strand is read 3’  5’ • So copied strand is synth’d 5’  3’ • Complementary strands are antiparallel • DNA double helix must be unwound in both • Topoisomerases impt to relieve tension on helix in both

6. Transcription DNARNA Different than Repl’n DNADNA • Amt DNA copied • Repl’n: entire chromosome copied • Both strands of dbl helix copied • Transcr’n: only 1 gene (part of chromosome) from 1 strand of double helix is copied •  Single strand mRNA • BUT gene is copied more than once • Yields many transcripts of same gene

7. Each strand of DNA being transcribed has different name • RNA transcribed from DNA template strand (26-2) • Complementary strand of dbl helix is called DNA nontemplate strand or coding strand • This strand has same seq as RNA transcript, except for one difference • How is it different from transcript??

8. Transcription Is Different – cont’d • Origin • Repl’n: one origin in E. coli • What’s that called? • Transcr’n: Enz’s/prot’s must know where along length of DNA to begin copying/stop copying • Polymerase • Repl’n: DNA polymerase • Several types w/ varied subunits • Has proofreading ability • Requires primer • Elongation up to 1,000 nucleotides/sec

9. Transcr’n Is Different -- cont’d • Polymerase – cont’d • Transcr’n: RNA polymerase (26-4) • 1 complex w/ 6 subunits • Called “holoenzyme” • 1 subunit (s) directs rest of enz to site of initiation of transcr’n

10. Transcription Is Different – cont’d • Transcr’n: RNA polymerase – cont’d • No proofreading • No primer needed • Begins mRNA w/ GTP or ATP • Elongation ~50-90 nucleotides/sec • Unwinding • Repl’n: helicases are used • Transcr’n: RNA polymerase keeps ~17 bps unwound

11. E. coli Promoter Region • DNA seq @ which transcr’n apparatus comes together to begin copying the gene • So each gene has a promoter • Consensus DNA seq’s -- highly conserved in both seq and location (26-5)

12. E. coli Promoter – cont’d • Consensus DNA seq’s – cont’d • +1 base = first nucleotide to be transcribed • Usually a purine • What are the purines?? • -10 region (toward 3’ end of template strand) = 6 nucleotide seq w/ consensus TATAAT • Spacer = ~16-18 nucleotides • -35 region = 6 nucleotide seq w/ consensus TTGACA • -40  -60 region = AT-rich region = Up-stream Promoter (UP element)

13. Fig.26-5

14. E. coli Promoter – cont’d • When pattern met exactly • RNA polymerase recognizes most efficiently • Get rapid transcription • When pattern varies from consensus sequences • Takes longer for RNA polymerase to recognize promoter • Get longer time of transcription

15. Initiation of Transcr’n in E. coli (26-6) • s subunit of RNA polymerase searches for promoter region • Scans ~2000 nucleotides/~ 3 sec along template strand • Holoenzyme binds at promoter region  “closed complex” • DNA bound to holoenzyme is intact • About 15 bps unwound @ -10 region  “open complex” • Probably conform’l changes in polymerase enz assist in “opening”

16. Initiation of Transcr’n – cont’d • Now transcription initiated w/ nucleotides matched to template strand, added to polymer • After ~8-9 nucleotides added, s subunit dissociates • Can scan another region to find another promoter

17. Initiation of Transcr’n – cont’d • Regulation of transcr’n • Strength of consensus at promoter region, as mentioned • Some polymerases have >1 s subunit • Cell stress  use of diff s subunit, specific for partic promoters needed to alleviate specific stresses

18. Initiation of Transcr’n – cont’d • Regulation of transcr’n – cont’d • Proteins may bind DNA seq’s in/around promoter • Some attract RNA polymerase to promoter region • So activate transcr’n of these genes • Some block RNA polymerase from binding @ promoter • Called “repressors” • So repress transcr’n of these genes • Proteins respond to metab, repro, stress conditions w/in the cell • Conditions may require much peptide or depletion of peptide • REMEMBER: Mech’s by cell to regulate glycolysis/metab??

19. Elongation of Transcr’n in E. coli • Holoenzyme free to move along template chain • Freer w/ dissoc’n of s subunit • Forms “transcription bubble” • Contains holoenzyme, template strand, new RNA strand

20. Elongation of Transcr’n – cont’d • New RNA strand “transiently” base-paired to template DNA strand (26-1) •  DNA-RNA hybrid

21. Elongation of Transcr’n – cont’d • DNA helix rewinds behind transcription bubble (26-1)

22. Elongation of Transcr’n – cont’d • Error rate in transcr’n ~1/105 bases added • Much higher than in repl’n • Acceptable • Cell will make many transcripts of same gene • Most  proper (active) peptides • Some  improper peptides that can be accommodated by cell • What if template strand were mutated?

23. Termination of Transcr’n in E. coli • Need RNA polymerase to be processive • If falls off, must re-start @ promoter • What might happen in cell if problem w/ RNA polymerase processivity? • BUT may pause @ certain template strand seq’s • Some template strand seq’s cause RNA polymerase to stop

24. Termination of Transcr’n – cont’d • Two types of termination in E. coli • Rho (r) independent (26-7) • Template seq  RNA transcript w/ self-complementary nucleotides • ~ 15-20 nucleotides • G-C rich, followed by A-T rich regions • Transcript forms stable hairpin loop • Template has string of A nucleotides  string of U nucleotides in transcript @ 3’ end • Causes RNA polymerase to pause

25. Termination of Transcr’n – cont’d • Rho (r) independent – cont’d • Stable hairpin of transcript, followed by relatively unstable A-U pairings of DNA-RNA hybrid  RNA transcript dissociates

26. Termination of Transcr’n – cont’d • Rho (r) independent -- cont’d • Polymerase dissociates • DNA helix reanneals, rewinds • r dependent • r = protein = termination factor • Binds RNA transcript @ partic binding sites • Moves along new transcript 5’  3’ to transcr’n bubble • Finds elongation paused • Disrupts DNA-RNA hybrid • Mechanism unknown • Has ATP hydrolysis ability

27. Prokaryote Transcription • Prokaryote chromosome in cytoplasm • No organized nucleus • Prokaryote chromosome simple • mRNA transcr’d directly from DNA seq • No introns/exons; “junk” DNA; etc. • As mRNA synth’d, almost immediately translated  peptide

28. EukaryoteTranscription • More complex, less understood • 3 RNA polymerases – I, II, III • Each w/ specific function • Each binds diff promoter seq • RNA Polymerase I • Transcribes some rRNA’s • RNA Polymerase III • Transcribes tRNA’s and rRNA’s

29. EukaryoteTranscription – cont’d • RNA Polymerase II • Transcribes mRNA (so most impt to transcr’n process) • Many subunits • Recognizes many promoters • Requires transcription factors

30. EukaryoteTranscription – cont’d • Transcription factors (Table 26-1) • Proteins • Modulate binding of RNA polymerase II to promoter region • Complex w/ RNA polymerase  proper binding to template, proper elongation (26-9)

32. Fig.26-9

33. EukaryoteTranscription – cont’d Takes place in nucleus • mRNA transcript  cytoplasm for translation • For peptides to be used outside the nucleus • REMEMBER: nuclear membr has pores • Euk genes complicated • REMEMBER: introns/exons, “junk?” DNA • Polymerase doesn’t seem to distinguish • Euk DNA transcr’d directly  mRNA • Yields a primary transcript directly reflecting entire gene and any introns/junk

34. EukaryoteTranscription – cont’d • For functioning peptide, intron seq’s excised before translation • So primary mRNA transcripts are spliced, rejoined • Through transesterification reaction (26-13) • Similar to topoisomerase mechanism

35. EukaryoteTranscription – cont’d Euk genes complicated – cont’d • Intron seq’s excised – cont’d • Most nuclear mRNA’s spliced by specialized RNA-protein complexes • snRNP’s = small nuclear RiboNucleoProteins • About 5 RNA’s + 50 prot’s complex  spliceosome (26-16) • Get “lariat” structure of intron seq nucleotides • Get attack by exon –OH end  phosphate @ other exon end

36. Fig.26-16

37. EukaryoteTranscription – cont’d • Euk mRNA’s also further modified at ends • 5’ cap • 7-Methylguanosine added @ 5’ end • Get 5’, 5’-triphosphate linkage (26-18) • May be impt in initiation of translation

38. EukaryoteTranscription – cont’d • Euk mRNA’s also further modified at ends – cont’d • 3’ polyA tail • 80-250 adenylate residues (26-19) • May stabilize mRNA against enz destruction

39. EukaryoteTranscription – cont’d • Final transcript = mature mRNA (26-20)

40. Fig.26-11

41. Inhibition of Transcription by Antibiotics • Actinomycin D (26-10) • Planar, non-polar • Intercalates between nucleotide bases of DNA • Esp. between G-C’s in G-C rich seq’s • Now polymerase can’t move along DNA template

42. Fig.26-10