RNA synthesis/Transcription IIBiochemistry 302February 6, 2006Biochemical evidence of RNAP binding to lac promoter: Footprint analysisLehninger Principles of Biochemistry, 4th ed., Ch 26Nuclease protection assayE. coli RNAP binding to T7 A3 promoter based on chemical modification• Susceptibility of guanine residues to DMS (dimethylsulfate)-induced methylation (± RNAP): ↓methylation w/RNAP,↑methylation w/RNAP, methylation prevents RNAP binding• Susceptibility of phosphate oxygens to ENU (ethylnitrosourea) modification (± RNAP): ♦• Note that the two conserved regions of the promoter are exactly two helix turns apart. What does this mean? (RNAP binds to one side of duplex DNA.)Fig. 26-14Transcription 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.σ factors: regulatory proteins 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.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 1strNTPPu5: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. 26-6Putative structure of E. coli core RNA polymerase during elongation phaseβ and β′ subunits: light gray and white, α subunits shades of red, ω subunit on other side not visible. Active site is a cleft between β and β′ subunits. 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), RNA/DNA heteroduplexor hybrid region (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. 26-9Consequences of RNAP movement:positive & negative DNA supercoilingLehninger Principles of Biochemistry, 4th ed., Ch 26But 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 or mismatched bp).• Resolution of pause sites may involve backtracking to create a free RNA 3′ end which is displaced from the active site. • GreB (or GreA) binds transiently to RNAP active site to stimulate its intrinsic transcript hydrolysis activity creating a new base-paired 3′ end.Fig. 26-10“backtracked” RNAPDonation of catalytic residues to RNAP by GreB (RNAP in hydrolysis mode)Sosunova et al. PNAS100:15469, 2003GreB turned 120°relative to RNAP β′Evolutionary conservation of “tunable”RNA polymerase active siteN. Opalka et al. Cell 114:335-345, 2003…S. Darst labH. Kettenberger et al. Cell 114:347-357, 2003…P. Cramer labFigure from editorial by R. ConawayTermination 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 sequence-dependent) terminationa: RNAP pauses when it reaches G:C sequence that enzyme finds hard to unwind.b: Pausing allows time for self-complementary regions of RNA transcript to bp. This displaces some RNA from DNA & RNAP RBS.c: Unstable A-U bonds cannot hold 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. 26-15Rho-dependent termination…less frequent and more complex1: Rho (ρ) protein binds as a homohexamer to RNA via a CA-rich recognition 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. NusA = Nutilization substanceFig. 26-16Repertoire of eukaryotic RNAPs• Pol I (13 subunits, ~600 kDa)– Nucleolar enzyme– Synthesis of pre-rRNA (45S RNA transcript →18S, 5.8S, 23 S rRNAs, mammals)• Pol II (12 subunits, ~500 kDa)– Nuclear enzyme– Synthesis of pre-mRNA and some snRNAs– Very sensitive to α-amanitin• Pol III (14 subunits, ~700 kDa)– Synthesis of pre-tRNA– Synthesis of 5S rRNA and remaining snRNAs• Mitochondrial RNAP• Chloroplast RNAPFig. 1.11Features of promoter & regulatory regions of genes transcribed by RNAPs• RNAP I – Transcribes multiple, tandemly arranged copies of gene encoding large precursor pre-rRNA (45S in E. coli)– Core promoter and proximal upstream elements that vary greatly from species to species– Distal upstream elements• RNAP II– Transcribes mainly protein-coding genes but some snRNAgenes as well – Conserved TATA box and Inr elements (general factors)– Gene-specific upstream elements & enhancers (regulatory factor binding sites)• RNAP III – Transcribes 5S rRNA and tRNAs, some snRNAs and