RNA synthesis/Transcription IBiochemistry 302RNA metabolism: major and minor classes of RNAOverview of RNA polymerasesFeatures of RNA vs DNA synthesisAnatomy,chemistry, and nomenclature of RNAP-mediated transcription in E. coliBiochemical features of E. coli RNAP? factors: regulatory factors which direct transcription of certain genesTranscription like replication can be construed to occur in distinct stepsTranscription initiation: key role of the gene promoterSequence conservation of core promoter elements (RNAP-?70)Genetic evidence for functional importance of core promoter elements(naturally-occurring and site-directed mutations)Biochemical evidence of RNAP binding to lac promoter: Footprint analysisPutative structure of E. coli core RNA polymerase during elongation phaseTranscription elongation: a detailed viewBut elongation of ternary complex often proceeds discontinuously….Donation of catalytic residues to RNAP by GreB (RNAP in hydrolysis mode)Termination of transcription: another process controlled by signals in DNARho factor-independent (or sequence-dependent) terminationRho-dependent termination…less frequent and more complexRNA synthesis/Transcription IBiochemistry 302February 4, 2005Bob KelmRNA metabolism: major and minor classes of RNA• Messenger RNA (mRNA)– Relatively short half-life (∼3 min in E. coli, ∼30 min in eukaryotic cells)• Ribosomal RNA (rRNA)– Major structural components of the ribosome• Transfer RNA (tRNA)– Adaptor molecules allowing physical linkage between mRNA and amino acids • “Small” RNAs – snRNAs (splicing)– Components of RNP enzymes (e.g. RNase P)– miRNAs (micro RNAs involved in PTGS)Overview of RNA polymerases• Prokaryotes– Single processive RNA polymerase (technically, primase is a RNAP too).– Inhibited by rifampicin (binds RNAP β subunit & blocks path of RNA chain elongation)• Eukaryotes– Three processive RNAPs– Differential sensitivity to inhibition by α-amanitin• RNA Pol I (resistant) → rRNA • RNA Pol II (low conc) → mRNA• RNA Pol III (high conc) → tRNA plus 5S rRNAFig. 26.4Note: α-amanitin, a non-competitive inhibitor, stops the translocation of RNAP along the DNA template after the formation of the first phospho-diester bond.Features of RNA vs DNA synthesis• Similarities to DNA synthesis– Synthesis of ribonucleotide chain is template-dependent.– Substrates are nucleoside triphosphates (rNTPs).– Direction of chain growth is 5′→3′.– Same chemical mechanism applies (base-pairing of incoming rNTP, 3′ OH attack, loss of PPi).– Highly processive enzyme• Differences from DNA synthesis– One DNA strand is transcribed per gene w/o a primer.– Only certain genes are transcribed at any given time.– Kinetics favor “slow” transcription of multiple genes. (Vmax ∼50 nt/s for RNA Pol vs ∼103/s for DNA Pol III; ∼3000 RNA Pol/cell vs ∼10 DNA Pol III complexes/cell)– Less accurate ∼10-5vs 10-10– Cofactor-mediated proofreadingAnatomy,chemistry, and nomenclature of RNAP-mediated transcription in E. coli~17 bpNTP~35 bp for RNAP “footprint”Lehninger Principles of Biochemistry, 4th ed., Ch 26Biochemical features of E. coli RNAP Core RNAP*contains part of active siteholoenzyme assemblysliding clamp• 450 kDa enzyme containing six subunits• Two Mg2+and one Zn2+required (chemistry and clamping)• No independent 3′→5′ exonuclease activity but may have kinetic proofreading capabilities• Two binding sites for ribonucleotides – Initiation site binds only purine rNTPs (GTP or ATP) with Kd= 100 µM…most mRNAs start with purine on 5′ end.– Elongation site binds any of 4 rNTPs with Kd= 10 µM.σ factors: regulatory factors which direct transcription of certain genes• Assist RNAP in binding DNA at the proper site for initiation of transcription – the promoter. • Different sigma factors orchestrate transcription of different classes of genes. – Heat shock (σ35) – Other stress responses– Metabolic enzymes (σ70, most abundant)• Not required for core RNA polymerase activity.Transcription like replication can be construed to occur in distinct steps• Initiation (requires special signals)– RNAP recognizes the promoter, binds to DNA, and starts transcription.– Highly regulated• Elongation– RNAP tracks down the length of the gene synthesizing RNA along the way. • Termination (requires special signals)– Transcription stops then RNAP and the nascent mRNA dissociate.Features of initiation phase in E. coli 1:RNAP binding and sliding (electrostatic interaction)2:Formation of closed complex (–55 to –5, Ka∼107-108 M−1,T½~10 s)3:Formation of open complex (–10 to –1, Ka∼1012 M−1, T½~15s to 20 min), temp-dependent, stable4:Mg2+-dependent conformational change (–12 to +2), add 1stnt5:Promoter clearance: RNAP moves away from promoter6:Release of σ after first 8-9 nts & continuation of elongation (now cannot be inhibited by rifampicin)7,8:Pausing → TerminationSignal for specific DNA-binding seen by σ factorFig. 26-6Transcription initiation: key role of the gene promoter• RNAP binding sequence: −70 to +30 in E. coli• DNA sequence specifying start site and basal rate of transcription– Constitutive: Specify that a gene product will be transcribed at a constant rate (e.g. genes involved in metabolic control)– Inducible or regulated: Specify transcription of certain genes in response to external signals (requires additional protein-DNA interactions)• Promoter recognition by RNAP: rate limiting for transcription (structure → frequency of initiation)• Promoters: exhibit certain consensus sequencesSequence conservation of core promoter elements (RNAP-σ70)•Variations in sequence and core element position account for differences in frequency of initiation.• 1975, David Pribnow and Heinz Schaller independently defined consensus promoter sequences, the –10 region or Pribnow box (TATAAT) and the –35 region (TTGACA). • Among 114 E. coli promoters studied, 6/12 nucleotides in the two consensus elements found in 75% of promoters. Transcription start siteFig. 26-11Genetic evidence for functional importance of core promoter elements(naturally-occurring and site-directed mutations)• The more closely core elements resemble the consensus, the more efficient the promoter at initiating transcription.• ↑Mutations: those toward the consensus sequence.• ↓Mutations: those away from the consensus