rna synthesis/transcription i biochemistry 302 february...
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RNA synthesis/Transcription IBiochemistry 302
February 3, 2006
Information readout/transcription: 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: (rNMP)n + rNTP →(rNMP)n+1 + PPi
– Inhibited by rifamycins• Steric mechanism: binds
RNAP β subunit & blocks elongation of RNA chain beyond 3 nucleotides
• Allosteric mechanism: removal of catalytic Mg2+
from active site (new data)• 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 rRNANote: α-amanitin, a non-competitive inhibitor, stops the translocation of RNAP along the DNA template after the formation of the first phospho-diester bond.
Fig. 26.4
Features of RNA synthesis compared to DNA synthesis• Similarities
– Synthesis of ribonucleotide chain is template-dependent.– Direction of chain growth is 5′→3′.– Same chemical mechanism applies (base-pairing of incoming
rNTP, 3′ OH attack, loss of PPi).– RNAP: highly processive enzyme
• Differences– One DNA strand is transcribed per gene w/o a primer.– Substrates are ribonucleoside triphosphates (rNTPs).– Only certain genes are transcribed at any given time.– Kinetics favor “slow” transcription of multiple genes.
• Vmax ∼50 nt/s for RNAP vs ∼103 nt/s for DNAP III• ∼3000 RNAP/cell vs ∼10 DNAP III complexes/cell
– Less accurate ∼1/105 vs 1/1010
– Proofreading is cofactor-dependent.– Synthesis coincides with localized RNAP-mediated unwinding of
DNA template.
Anatomy, chemistry, and nomenclature of RNAP-mediated transcription in E. coli
~35 bp for RNAP “footprint”
~17 bp
Lehninger Principles of Biochemistry, 4th ed., Ch 26
rNTP
Asp460, Asp462, Asp464 in β′ subunit
Structure/Function of E. coli RNAP
• 450 kDa holoenzyme containing six subunits• Two Mg2+ and one Zn2+ required (catalysis 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.
Core RNAP
*
contains part of active site
holoenzyme assembly
part of active site & sliding clamp
Transcription initiation: key role of the gene promoter• RNAP binding site in a gene: −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 core consensus sequences
Sequence conservation of corepromoter elements (RNAP-σ70)
• 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 seen in 75% of promoters.
• Variations in sequence and core element position account for differences in frequency of initiation.
Transcription start siteFig. 26-11
Positional conservation and functional importance of core promoter elements
• The more closely core elements resemble the consensus, the more efficient (or stronger) 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
Yellow: ~75% high conservationBlue: ~50-75% moderate conservationPurple: ~40-50% weak conservation
Naturally-occurring and site-directed mutations that affect promoter strength localize to nucleotides comprising the -35 and -10 core elements.
Biochemical evidence of RNAP binding to lac promoter: Footprint analysis
Lehninger Principles of Biochemistry, 4th ed., Ch 26
Nuclease protection assay
E. 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. 26-14
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.
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, stable
4:Mg2+-dependent conformational change (–12 to +2), add 1st rNTPPu
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)
7,8:Pausing → Termination
Signal for specific DNA-binding seen by σ factor
Fig. 26-6
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. Active site is a cleft between β and β′ subunits.
Note circuitous route taken by the DNA and RNA through the complex.
Consequences of RNAP movement:positive & negative DNA supercoiling
Lehninger Principles of Biochemistry, 4th ed., Ch 26