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Transcript
Chapter 21 (part 1) Transcription Central Dogma RNA Content of E. coli Cells type Steady State Levels Synthetic Capacity Stability rRNA 83% 58% High tRNA 14% 10% High mRNA 3% 32% Very Low E. Coli RNA Polymerase • RNA polymerase core enzyme is a multimeric protein a2,b, b’, w • The b’ subunit is involved in DNA binding • The b subunit contains the polymerase active site • The a subunit acts as scaffold on which the other subunits assemble. • Also requires s-factor for initiation –forms holo enzyme complex Site of DNA binding and RNA polymerization General Gene Structure 5’ Promoter Transcribed region terminator 3’ Gene Promoters Other s-Factors • Standard genes – s70 • Nitrogen regulated genes – s54 • Heat shock regulated genes – s32 Transcriptional Initiation Closed complex Open complex Primer formation Disassociation of s-factor Pausing induces termination 3’end tends to be AU rich easily to disrupt during pausing. Leads to disassembly of RNA polymerase complex Rho Dependent Termination • rho is an ATPdependent helicase • it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain Eukaryotic RNA Polymerases type Location Products RNA polymerase I Nucleolus rRNA RNA polymerase II Nucleoplasm mRNA RNA polymerase rRNA, tRNA, Nucleoplasm III others Mitochondrial RNA Mitochondrial gene Mitochondria polymerase transcripts Chloroplast RNA polymerase Chloroplast Chloroplast gene transcripts Eukaryotic RNA Polymerases • RNA polymerase I, II, and III • All 3 are big, multimeric proteins (500-700 kD) • All have 2 large subunits with sequences similar to b and b' in E.coli RNA polymerase, so catalytic site may be conserved Eukaryotic Gene Promoters • Contain AT rich concensus sequence located –19 to –27 bp from transcription start (TATA box) • Site where RNA polymerase II binds Transcription Factors • TFAIIA, TFAIIB – components of RNA polymerase II holoenzyme complex • TFIID – Initiation factor, contains TATA binding protein (TBP) subunit. TATA box recognition. • TFIIF – (RAP30/74) decrease affinity to nonpromoter DNA
