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Transcription

Transcription. Chapter 8. The Problem. Information must be transcribed from DNA in order function further. REMEMBER: DNA RNAProtein. Tanscription in Prokaryotes. Polymerization catalyzed by RNA polymerase Can initiate synthesis Uses rNTPs Requires a template

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  1. Transcription Chapter 8

  2. The Problem • Information must be transcribed from DNA in order function further. • REMEMBER: • DNARNAProtein

  3. Tanscription in Prokaryotes • Polymerization catalyzed by RNA polymerase • Can initiate synthesis • Uses rNTPs • Requires a template • Unwinds and rewinds DNA • 4 stages • Recognition and binding • Initiation • Elongation • Termination and release

  4. RNA Polymerase • 5 subunits, 449 kd (~1/2 size of DNA pol III) • Core enzyme • 2  subunits---hold enzyme together • --- links nucleotides together • ’---binds templates • ---recognition • Holoenzyme= Core + sigma

  5. RNA Polymerase Features • Starts at a promoter sequence, ends at termination signal • Proceeds in 5’ to 3’ direction • Forms a temporary DNA:RNA hybrid • Has complete processivity

  6. RNA Polymerase • X-ray studies reveal a “hand” • Core enzyme closed • Holoenzyme open • Suggested mechanism • NOTE: when sigma unattached, hand is closed • RNA polymerase stays on DNA until termination.

  7. Recognition • Template strand • Coding strand • Promoters • Binding sites for RNA pol on template strand • ~40 bp of specific sequences with a specific order and distance between them. • Core promoter elements for E. coli • -10 box (Pribnow box) • -35 box • Numbers refer to distance from transcription start site

  8. Template and Coding Strands Sense (+) strand DNA coding strand Non-template strand DNA template strand antisense (-) strand 5’–TCAGCTCGCTGCTAATGGCC–3’ 3’–AGTCGAGCGACGATTACCGG–5’ transcription 5’–UCAGCUCGCUGCUAAUGGCC–3’ RNA transcript

  9. Consensus sequences Typical Prokaryote Promoter • Pribnow box located at –10 (6-7bp) • -35 sequence ~(6bp) • Consensus sequences: Strongest promoters match consensus • Up mutation: mutation that makes promoter more like consensus • Down Mutation: virtually any mutation that alters a match with the consensus

  10. In Addition to Core Promoter Elements • UP (upstream promoter) elements • Ex. E. coli rRNA genes • Gene activator proteins • Facilitate recognition of weak promoter • E. coli can regulate gene expression in many ways

  11. Stages of Transcription • Template recognition • RNA pol binds to DNA • DNA unwound • Initiation • Elongation • RNA pol moves and synthesizes RNA • Unwound region moves • Termination • RNA pol reaches end • RNA pol and RNA released • DNA duplex reforms

  12. Transcription Initiation • Steps • Formation of closed promoter (binary) complex • Formation of open promoter complex • Ternary complex (RNA, DNA, and enzyme), abortive initiation • Promoter clearance (elongation ternary complex) • First rnt becomes unpaired • Polymerase loses sigma • NusA binds • Ribonucleotides added to 3’ end

  13. Back • Holoenzyme • Core +  • Closed (Promoter) Binary Complex • Open binary complex • Ternary complex • Promoter clearance

  14. Sigma () Factor • Essential for recognition of promoter • Stimulates transcription • Combines with holoenzyme • “open hand” conformation • Positions enzyme over promoter • Does NOT stimulate elongation • Falls off after 4-9 nt incorporated • “Hand” closes

  15. Variation in Sigma • Variation in promoter sequence affects strength of promoter • Sigmas also show variability • Much less conserved than other RNA pol subunits • Several variants within a single cell. EX: • E. coli has 7 sigmas • B. subtilis has 10 sigmas • Different  respond to different promoters

  16. Sigma Variability in E. coli • Sigma70 (-35)TTGACA (-10)TATAAT • Primary sigma factor, or housekeeping sigma factor. • Sigma54 (-35)CTGGCAC (-10)TTGCA • alternative sigma factor involved in transcribing nitrogen-regulated genes (among others). • Sigma32 (-35)TNNCNCNCTTGAA (-10)CCCATNT • heat shock factor involved in activation of genes after heat shock. • POINT: gives E. coli flexibility in responding to different conditions

  17. Sigma and Phage SP01 • Early promoter—recognized by bacterial sigma factor. Transcription includes product, gp28. • gp28 recognizes a phage promoter for expression of mid-stage genes, including • gp33/34, which recognizes promoters for late gene expression.

  18. Promoter Clearance and Elongation • Occurs after 4- 10 nt are added • First rnt becomes unpaired from antisense (template) strand.DNA strands re-anneal • Polymerase loses sigma, sigma recycled • Result “Closed hand” surrounds DNA • NusA binds to core polymerase • As each nt added to 3’, another is melted from 5’, allowing DNA to re-anneal. • RNA pol/NusA complex stays on until termination. Rate=20-50nt/second.

  19. Termination • Occurs at specific sites on template strand called Terminators • Two types of termination • Intrinsic terminators • Rho () dependent treminators • Sequences required for termination are in transcript • Variation in efficiencies.

  20. Intrinsic Terminators • DNA template contains inverted repeats (G-C rich) • Can form hairpins • 6 to 8 A sequence on the DNA template that codes for U • Consequences of poly-U:poly-A stretch? Coding strand

  21. Intrinsic Termination • RNA pol passes over inverted repeats • Hairpins begin to form in the transcript • Poly-U:poly-A stretch melts • RNA pol and transcript fall off UUUUU

  22. Rho () Dependent Terminators • rho factor is ATP dependent helicase • catalyses unwinding of RNA: DNA hybrid

  23. (17 bp) Rho Dependent Termination • rho factor is ATP dependent helicase • catalyzes unwinding of RNA: DNA hybrid • 50~90 nucleotides/sec

  24. Rho: Mechanism hexamer • Rho binds to transcript at  loading site (up stream of terminator) • Hairpin forms, pol stalls • Rho helicase releases transcript and causes termination

  25. Abortive initiation, elongation

  26. mRNA • Function—Transcribe message from DNA to protein synthesis machinery • Codons • Bacterial—polycistronic • Eukaryotic– monocistronic • Leader sequence—non-translated at 5’ end • May contain a regulatory region (attenuator) • Also untranslated regions at 3’ end. • Spacers (untranslated intercistronic sequences) • Prokaryotic mRNA—short lived • Eukaryotic mRNA-can be long lived

  27. Stable RNA • rRNA -Structural component of ribosomes • tRNA-Adaptors, carry aa to ribosome • Synthesis • Promoter and terminator • Post-transcriptional modification (RNA processing) • Evidence • Both have 5’ monophospates • Both much smaller than primary transcript • tRNA has unusual bases. EX pseudouridine

  28. tRNA and rRNA Processing • Both are excised from large primary transcripts • 1º transcript may contain several tRNA molecules, tRNA and rRNA • rRNAs simply excised from larger transcript • tRNAs modified extensively 5. Base modifications

  29. Examples of Covalent Modification of Nucleotides in tRNA 7-Methylguanylate (m7G) Inosinate (I) N6-Methyladenylate (m6A) N6-Isopentenyladenylate (i6A) Uridylate 5-oxyacetic acid (cmo5U) Dihydrouridylate (D) Pseudouridylate (Ψ) (ribose at C-5) 3-Methylcytidylate (m3C) Modifications are shown in blue. 5-Methylcytidylate (m5C) 2’-O-Methylated nucleotide (Nm)

  30. Eukaryotic Transcription • Regulation very complex • Three different pols • Distinguished by -amanitin sensitivity • Pol I—rRNA, least sensitive • Pol II– mRNA, most sensitive • Pol III– tRNA and 5R RNA moderately sensitive • Each polymerase recognizes a distinct promoter

  31. Eukaryotic RNA Polymerases

  32. Eukaryotic Polymerase I Promoters • RNA Polymerase I • Transcribes rRNA • Sequence not well conserved • Two elements • Core element- surrounds the transcription start site (-45 to + 20) • Upstream control element- between -156 and -107 upstream • Spacing affects strength of transcription

  33. Eukaryotic Polymerase II Promoters • Much more complicated • Two parts • Core promoter • Upstream element • Core promoter • TATA box at ~-30 bases • Initiator—on the transcription start site • Downstream element-further downstream • Many natural promoters lack recognizable versions of one or more of these sequences

  34. TATA-less Promoters • Some genes transcribed by RNA pol II lack the TATA box • Two types: • Housekeeping genes ( expressed constitutively). EX Nucleotide synthesis genes • Developmentally regulated genes. EX Homeotic genes that control fruit fly development. • Specialized (luxury) genes that encode cell-type specific proteins usually have a TATA-box

  35. mRNA Processing in Eukaryotes • Primary transcript much larger than finished product • Precursor and partially processed RNA called heterogeneous nuclear RNA (hnRNA) • Processing occurs in nucleus • Splicing • Capping • Polyadenylation

  36. Capping mRNA • 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. • Guanosine cap is methylated. • First and second nucleosides in mRNA may be methylated BACK

  37. Polyadenylation • Polyadenylation occurs on the 3’ end of virtually all eukaryotic mRNAs. • Occurs after capping • Catalyzed by polyadenylate polymerase • Polyadenylation associated with mRNA half-life • Histones not polyadenylated

  38. Introns and Exons • Introns--Untranslated intervening sequences in mRNA • Exons– Translated sequences • Process-RNA splicing • Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete

  39. Splicing Overview • Occurs in the nucleus • hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP) • Primary transcripts assembled into hnRNP • Splicing occurs on spliceosomes consist of • Small nuclear ribonucleoproteins (SnRNPs) • components of spliceosomes • Contain small nuclear RNA (snRNA) • Many types of snRNA with different functions in the splicing process

  40. Spliceosome

  41. Splice Site Recognition • Introns contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule) • Recognized by small nuclear ribonucleoprotein particles (snRNPs) that catalyze the cutting and splicing reactions. • Internal intron sequences are highly variable even between closely related homologous genes. • Alternative splicing allows different proteins from a single original transcript

  42. Simplified Splicing Mechanism

  43. Close-up of Internal A

  44. Alternative Splicing I • Exon removed with intron

  45. Alternative Splicing II • Multiple 3’ cleavage sites • EX. AG found at 5’ end of exon 2 and inside exon 2

  46. RNA pol III • Precursors to tRNAs,5SrRNA, other small RNAs • Promoter Type I • Lies completely within the transcribed region • 5SrRNA promoter split into 3 parts • tRNA promoters split into two parts • Polymerase II-like promoters • EX. snRNA • Lack internal promoter • Resembles pol II promoter in both sequence and position

  47. DNAse Footprinting • Use: promoter ID • End Label template strand • Add DNA binding protein • Digest with DNAse I • Remove protein • Separate on gel Protected region

  48. siRNA and microRNA

  49. Maxam-Gilbert Sequencing • Prep ssDNA • End label • Treat with different reagents specific for each nt

  50. RNA Splicing RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a gene Excision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons)

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