Transcription in prokaryotes

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Transcription in prokaryotes
2012-05-12 07:51:22
transcription RNA RNAP prokarotic prokaryote prokaryotes

Transcription in prokaryotes
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  1. End of RNA chain has?
    triphosphate group (5'pppN - N = A/G)
  2. Error frequency for RNAP
    1/10^4 nt
  3. 2 promoter consensus sequences in prokaryotes:
    • Pribnow box: TATAAT (coding (mRNA) side) in -10 region
    • -35 box: TTGACA
  4. Closed/Open complex?
    • Closed: Holoenzyme binds to promoter
    • Open: 2 strands of DNA are opened over a length of ~15bp and transcription starts
  5. Components of RNAP
    • α2, β, β', δ (holoenzyme)
    • δ induces a high affinity for promoters, suppresses random non-specific binding
    • β and β' form a clamp
  6. Termination of RNA synthesis
    • Rho-independent: GC rich hairpin, run of ~6 Us - hairpin stalls RNAP, low affinity for Us causes dissociation
    • Rho-dependent: uses Rho factor (a helicase that unwinds RNA-DNA or RNA-RNA using ATP), requires 50-90 nt C rich region
  7. sigma factors: most common, and heat shock
    • Common: δ70
    • Heat shock: δ32
  8. 3 ways to regulate gene expression in prokaryotes
    • Promoter efficiency
    • Sigma factors
    • Regulatory proteins: repressors usually block binding site, positive proteins provide extra recognition contacts
  9. Explain Lac Operon
    • lactose -> galactose + glucose uses β-galactosidase
    • β-galactosidase transcription is usually blocked by the Lac repressor
    • β-galactosides eg lactose or non-hydolysable analogue eg IPTG bind to the Lac repressor, so it cannot bind to DNA
    • RNAP can bind to DNA and initiate transcription
  10. Explain CAP
    • CAP is Catabolite Activator Protein
    • Activated by cAMP due to low glucose
    • Makes enzymes to digest other sugars eg lactose
  11. 2 small molecule inhibitors of RNA synthesis
    • Actinomycin D: intercalates between neighbouring GC pairs in dsDNA. Blocks all RNA synthesis immediately
    • Rifamycin: binds the β-subunit of RNAP. Blocks all bacterial RNA synthesis. Does not affect eukaryotic! Used to treat TB.