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RNA synthesis/Transcription I Biochemistry 302 February 4, 2005 Bob Kelm RNA 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 rRNA Fig. 26.4 Note: α-amanitin, a non-competitive inhibitor, stops the translocation of RNAP along the DNA template after the formation of the first phosphodiester 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 ∼10-5 vs 10-10 – Cofactor-mediated proofreading Anatomy,chemistry, and nomenclature of RNAP-mediated transcription in E. coli ~17 bp NTP ~35 bp for RNAP “footprint” Lehninger Principles of Biochemistry, 4th ed., Ch 26 Biochemical features of E. coli RNAP Core RNAP holoenzyme assembly contains part of active site * • • • • sliding 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) Signal for specific DNA-binding seen by σ factor 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, stable 4:Mg2+-dependent conformational change (–12 to +2), add 1st nt 5:Promoter clearance: RNAP moves away from promoter 6:Release of σ after first 8-9 nts & continuation of elongation (now cannot be inhibited by rifampicin) Fig. 26-6 7,8:Pausing → Termination Transcription 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 sequences Sequence 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. Fig. 26-11 Transcription start site Genetic 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 sequence. • Spacing (optimal 17 bp) between core consensus sequences is important. Fig. 26-12 Biochemical evidence of RNAP binding to lac promoter: Footprint analysis Lehninger Principles of Biochemistry, 4th ed., Ch 26 Putative structure of E. coli core RNA polymerase during elongation phase β and β′ subunits: light gray and white, α subunits shades of red, ω subunit on other side not visible. Note circuitous route taken by the DNA and RNA through the complex. Transcription elongation: a detailed view • Elongation complexes are stabilized by contact between specific regions/residues of β/β′ and the growing RNA chain (RBS), heteroduplex (HBS), or “downstream” DNA (DBS). • Core RNAP moves along the DNA template simultaneously unwinding DNA ahead and rewinding the template behind. Zn2+-binding domain of β′ subunit is the sliding clamp. RNAP activity requires Mg2+. Formation of 5′ RNA hairpin may be a signal for termination. Fig. 26-9 But elongation of ternary complex often proceeds discontinuously…. • Transcription “bubble” model implies continuous movement but RNAP may pause at difficult to “read” sites (e.g. high G/C content). “backtracked” RNAP Fig. 26-10 • Resolution of pause sites may involve backtracking to create a RNA 3′ end which is displaced from the active site. • GreA and GreB bind transiently to RNAP active site and stimulate its intrinsic transcript (i.e. RNA) hydrolysis activity creating a new base-paired 3′ end. Donation of catalytic residues to RNAP by GreB (RNAP in hydrolysis mode) Sosunova et al. PNAS 100:15469, 2003 GreB turned 120° relative to RNAP β′ Termination of transcription: another process controlled by signals in DNA • Termination signals are similar to signals that promote pausing – High G/C content (tend to form stem-loop structure) – Palindromic sequences that de-stabilize the DNA/RNA heteroduplex • Two types of termination mechanisms – Factor independent: Dyad symmetry followed by poly A sequence - intrastrand stem loop followed by rU:dA that destabilizes RNA/template – Factor (ρ, rho) dependent: Rho protein (RNA- dependent ATPase) destabilizes the RNA-DNA duplex. Rho factor-independent (or sequencedependent) termination a: RNAP pauses when it reaches G:C sequence that enzyme finds hard to unwind. b: Pausing allows time for selfcomplementary regions of RNA transcript to bp. This displaces some RNA from DNA & RNAP RBS. c: Unstable A-U bonds cannot hold weakened ternary complex (RNAP + RNA + DNA) together. RNAP and mRNA dissociate from the DNA template. Note: Actual mechanism is more complex and requires additional signals both 5′ and 3′. Fig. 26-15 Rho-dependent termination…less frequent and more complex 1: Rho (ρ) protein binds as a homohexamer to RNA at a CA-rich site (rut for rho utilization) near 3′ end and slides toward paused RNAP. 2: RNA-DNA helicase and ATPase activity of Rho unwinds RNA away from template DNA. 3: Template and transcript dissociate. Note: An additional protein, NusA, may be required for RNAP pausing. NusA binds to core RNAP after σ has dissociated. Fig. 26-16 NusA = N utilization substance