RNA synthesis/transcription I Biochemistry 302. February 6, 2004 Bob Kelm

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1 RNA synthesis/transcription I Biochemistry 302 February 6, 2004 Bob Kelm

2 Overview of RNA classes Messenger RNA (mrna) Encodes protein Relatively short half-life ( 3 min in E. coli, 30 min in eukaryotic cells) Ribosomal RNA (rrna) Comprise the major structural components of the ribosome. Transfer RNA (trna) Adaptor molecules allowing physical linkage between mrna and protein (amino acids) Small RNAs snrnas (splicing) Components of RNP enzymes (e.g. RNase P) mirnas (micro RNAs involved in PTGS)

3 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 Note: α-amanitin, a non-competitive inhibitor, stops the translocation of RNAP along the DNA template after the formation of the first phosphodiester bond.

4 Features of RNA 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 10 3 /s for DNA Pol III; 3000 RNA Pol/cell vs 10 DNA Pol III complexes/cell) Less accurate 10-5 vs 10-10

Conceptual view of RNA polymerase in action (elongation phase) Rewinding behind Unwinding ahead Nontemplate strand = coding strand Template strand = noncoding

5 Conceptual view of RNA polymerase in action (elongation phase) Rewinding behind Unwinding ahead Nontemplate strand = coding strand Template strand = noncoding strand Which way is the Polymerase moving? Fig Footprinting and RNase protection techniques are used to determine the length of DNA or RNA in contact with protein.

6 Biochemical features of E. coli RNA polymerase 450 kda enzyme containing five subunits β contains part of the active site. β responsible for DNA binding. α (dimer) mediates protein:protein interactions and allows complex to be assembled. ω is an accessory subunit whose function is unknown. σ mediates promoter recognition. Mg 2+ and Zn 2+ requiring (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 K d = 100 µm so most mrnas start with purine on 5 end. Elongation site binds any of 4 rntps with K d = 10 µm.

7 Subunit composition of E. coli RNA polymerase (450 kda holoenzyme) Core RNAP * Table 26-1 *Sigma factors play a key regulatory role by directing RNAP to bind DNA at the proper site for initiation of transcription the promoter. Different sigma factors orchestrate transcription of different classes of genes.

8 σ factors are true gene regulators in that they direct transcription of particular genes involved in. Sporulation Heat shock Other stress responses..but are not required for core RNA polymerase activity. σ 70 most abundant

9 Transcription like replication can be construed to occur in distinct phases Initiation (requires special signals) RNAP recognizes the promoter, binds to DNA, and starts transcription. 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.

Mechanism of RNA synthesis in E. coli (Note:Basic mechanics are similar in all organisms.) 1:RNAP binding and sliding (electrostatic interaction) Signal for specific DNA-binding seen by σ factor Fig.

10 Mechanism of RNA synthesis in E. coli (Note:Basic mechanics are similar in all organisms.) 1:RNAP binding and sliding (electrostatic interaction) Signal for specific DNA-binding seen by σ factor Fig :Formation of closed complex ( 55 to 5, K a M 1,T ½ ~10 s) 3:Formation of open complex ( 10 to 1, K a M 1, T ½ ~15s to 20 min), temp-dependent, stable 4:Mg 2+ -dependent isomerization ( 12 to +2) and addition of 1 st nt 5:RNAP starts moving away from promoter and synthesizing RNA 6:Release of sigma factor w/ continuation of elongation (now cannot be inhibited by rifampicin) 7,8:Pausing Termination

11 Transcription initiation: So what s a promoter. DNA sequence(s) specifying start of transcription different types 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) Basal: The minimal sequence within a constitutive or inducible promoter needed to initiate low-level but accurate transcription. Promoters have structure and consensus sequences can be identified

Conservation of E. coli promoter sequences Promoter recognition is rate limiting for transcription. Variations in promoter sequence account for differences in frequency of initiation.

12 Conservation of E. coli promoter sequences Promoter recognition is rate limiting for transcription. Variations in promoter sequence 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 sequences found in 75% of promoters. Fig Transcription start site

Genetic evidence for functionality of promoter sequences (naturally-occurring and site-directed mutations) The more closely a promoter resembles the consensus sequence, the more efficient it is at

13 Genetic evidence for functionality of promoter sequences (naturally-occurring and site-directed mutations) The more closely a promoter resembles the consensus sequence, the more efficient it is at initiating transcription. Mutations: those which make the promoter look more like the consensus. Mutations: those which move the promoter away from consensus. Fig Spacing (optimal 17 bp) between consensus sequences is important.

14 Biochemical evidence for E. coli RNA polymerase binding to T7 A3 promoter Fig 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?

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

15 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. Zn 2+ -binding domain of β subunit is the sliding clamp. RNAP activity requires Mg 2+. Formation of 5 RNA hairpin may be a signal for termination. Fig. 26-9

But elongation of ternary complex often proceeds discontinuously. backtracked RNAP Fig. 26-10 Transcription bubble model implies continuous movement but RNAP may pause at difficult to read sites (e.g. high G/C content).

16 But elongation of ternary complex often proceeds discontinuously. backtracked RNAP Fig 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.

17 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

18 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 (RNAdependent ATPase) destabilizes the RNA-DNA duplex.

19 Rho factor-independent (or sequencedependent) termination a: RNAP pauses when it reaches 1 st 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. Fig Note: Actual mechanism is more complex and requires additional signals both 5 and 3.

20 Rho-dependent termination less frequent and more complex 1: Rho (ρ) protein binds as a homohexamer to RNA at a C- rich site 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. (E. coli NusA must complex with viral N protein to allow viral gene expression: anti-termination.) Fig NusA = N utilization substance

RNA Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. RNA differs from DNA

RNA Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. RNA differs from DNA that it has a hydroxyl group at the 2 position of the