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 2 o 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 –PO 4 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 (  ) 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)  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,  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  subunit Cell stress  use of diff  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  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 ~1/10 5 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 (  ) 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 (  ) 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 (  ) independent -- cont’d Polymerase dissociates DNA helix reanneals, rewinds –  dependent  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