RNA synthesis/Transcription IBiochemistry 302

February 4, 2005Bob 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 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 ∼10-5 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

*

contains part of active site

holoenzyme assembly

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)

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)

7,8:Pausing → Termination

Signal for specific DNA-binding seen by σ factor

Fig. 26-6

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.

Transcription start siteFig. 26-11

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, ωsubunit 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).• 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.Fig. 26-10

“backtracked” RNAP

Donation of catalytic residues to RNAP by GreB (RNAP in hydrolysis mode)

Sosunova et al. PNAS100:15469, 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 sequence-dependent) termination

a: 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 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