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