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. 2015 Jul 31;427(15):2435-2450.
doi: 10.1016/j.jmb.2015.05.024. Epub 2015 Jun 6.

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

Emily F Ruff  1 , Amanda C Drennan  2 , Michael W Capp  3 , Mikaela A Poulos  2 , Irina Artsimovitch  4 , M Thomas Record Jr  5
Affiliations

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

Emily F Ruff et al. J Mol Biol. .

Abstract

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >10(5)-fold and >10(2)-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ(70) region 1.1 (σ1.1), β' jaw and β' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases -4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either β' jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.

Keywords: kinetics; mechanism; regulation; transcription initiation; σ(70) region 1.1.

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Figures

Figure 1
Figure 1
Minimal mechanism of formation and dissociation of stable OC (RPO) at λPR, showing steps that contribute to the observed kinetics (kobs, kd). Where the kinetics are single exponential [48], the initial steps (with composite equilibrium constants K1 or K3) rapidly equilibrate on the time scale of the subsequent rate-determining (k2 or k−2) step. For this situation, the dissociation rate constant kd is determined by the rate constant k−2 for DNA closing (I2 → I1) and the equilibrium constant K3 for stabilization of the open complex I2 to form longer-lived I3 and/or RPO complexes [3, 5]. Likewise, in RNAP excess, the rate constant for OC formation kobs is determined by the equilibrium constant K1 for formation of the ensemble of closed (I1) intermediates and the forward rate constant k2 of the isomerization step that includes DNA opening and converts this I1 ensemble to OCs [42, 43, 49, 50][49][50][50]. For these situations, kd = k−2/(1 + K3) (Equation 2) and kobs = k2 K1 [R]/(1 + K1 [R]).
Figure 2
Figure 2
E. coli RNAP holoenzyme (α2 ββ’ωσ70). A. View from above the active site cleft, showing RNAP as a van der Waals surface. Subunits: α2 : dark blue; β: yellow; β’: green; ω: light orange; σ residues 7-55: purple (note: 7 is the observed N-terminus); σ residues 56-98: red; σ residues 99-106: cyan; σ residues 107-613: light pink; β SI1 (residues 225-343): magenta; β’ SI3 (residues 943-1130): bright orange; β’ jaw deletion from [27] (residues 1149-1190): gray; active site Mg2+ position is indicated by an arrow. Structure adapted from PDB 4LK1 [16]. B. Same view as panel A, highlighting peptide backbone residues that contact NT strand (blue spheres) and T strand (black spheres) in a dinucleotide (GpA)-stabilized complex of Thermus thermophilus RNAP with a heteroduplex promoter fragment in which −12 to +2 (NT) and −4 to +2 (T) are ss DNA, with a 13 bp downstream duplex [34]. C. Orientation of σ70 region 1 (residues 7-106) in E. coli RNAP holoenzyme. Subunits are colored as in panel A, expanded 120%. Gaps in electron density observed within the flexible linker are labelled and represented as red dots. D. The sequences of λPR, T7A1, and rrnB P1 from the −35 element to base +2 are shown for comparison. The −10 and −35 elements are underlined; the transcription bubble bases (λPR : −11 to +2; T7A1: −12 to +2) are shown in blue; base +1A is shown in red.
Figure 3
Figure 3
Dissociation kinetic data for stable OCs formed by WT (white circles, black fit), △55 (purple circles and fit) and △98 (red circles and fit) RNAP, obtained by nitrocellulose filter binding. Nonlinear fits to Equation 1 are obtained from Igor Pro version 5.03 as described in Methods. A. Dissociation of λPR OCs at 37 °C. B. Data from A. replotted on a logarithmic time scale. C., D. Dissociation of T7A1 OCs at 37 °C, plotted and analyzed as in A. and B.
Figure 4
Figure 4
Kinetics of dissociation of T7A1-WT RNAP OCs. A. Nitrocellulose filter binding dissociation kinetic data for T7A1-WT RNAP complexes in TB (0.12 M KCl; white triangles with black fit) and after rapid mixing upshifts to 0.48 M (red triangles and line), 0.6 M (green triangles and line), 0.8 M (blue triangles and line), and 1.1 M KCl (purple triangles and line). B. Log-log dependences of dissociation rate constants kd for T7A1-WT RNAP (black triangles) OCs on KCl concentration at 37 °C. Data for λPR (white hourglasses), shown for comparison, are from [5]. At high salt concentration where K3 << 1, kd = k−2 which is found to be independent of salt concentration; these high-salt plateaus are shown by horizontal lines (T7A1: solid, average of four KCl points; λPR : dotted, from [5]).
Figure 5
Figure 5
Lifetimes and stabilizations of λPR and T7A1 OCs formed with RNAP variants. Comparison of lifetimes of stable OCs (1/kd; panel A) and initial OC I2 (1/k−2; panel B), and OC stabilization equilibrium constants (K3; panel C) for λPR (unshaded, left bar of each pair) and T7A1 (shaded, right bar of each pair) promoters and for WT RNAP, σ1.1 deletion △98, and β’ deletions △JAW and △SI3. All bar graphs use same log scale vertical axis, starting at the same point, so heights can be visually compared.
Figure 6
Figure 6
Comparison of lifetimes of stable OC (1/kd) and initial unstable OC (1/k−2) formed by λPR (blue) and T7A1 (red) promoters. The lifetime axis is on a logarithmic scale. Values for WT RNAP (first column) are compared with single deletions in σ1.1 (second column) and β’ DMEs (third column) and double deletions △55/△DME (fourth column) and △98/△DME (fifth column).
Figure 7
Figure 7
Permanganate footprinting of △98 and WT RNAP OCs at T7A1 and λPR. Positions of KMnO4-reactive thymines are labelled, as determined from A+G sequencing lanes (lanes A8, B7, C1, and D1). λPR NT strand footprints (panel A) of △98 RNAP open complexes are shown in the absence (lanes 4,6) and presence (lanes 5,7) of 20 μg/ml heparin; that of WT RNAP (+ 20 μg/ml heparin) is shown in lane 3. Free DNA controls are shown in the absence (lane 1) andpresence (lane 2) of 20 μg/ml heparin.λPR T strand footprints (panel B) of △98 RNAP OC are shown in the absence (lane 5) and presence (lane 6) of 20 μg/ml heparin; that of WT RNAP (+ 20 μg/ml heparin) is shown in lane 4. Free DNA controls are shown in the absence (lanes 1,3) and presence (lane 2) of 20 μg/ml heparin. T7A1 NT strand (panel C) and T strand (panel D) footprints of △98 RNAP OC (lanes C5, D5) and WT RNAP (lanes C4, D4) are compared to free DNA controls, exposed to KMnO4 (lanes C3, D3) or not exposed to KMnO4 (lanes C2, D2). Conditions: TB, 37 °C; 0.2 mM KMnO4, 10 s [1].
Figure 8
Figure 8
Interactions between RNAP elements and promoter DNA that define properties of the various OCs, compared with free RNAP (see Discussion). Note that only the DNA strands from ~ −10 to +20 are shown.
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References

    1. Ruff EF, Artsimovitch I, Record MT., Jr. Initial Events in Bacterial Transcription Initiation. Biomolecules. 2015;5:1035–62. - PMC - PubMed
    1. Saecker RM, Record MT, Jr., deHaseth PL. Mechanism of Bacterial Transcription Initiation: RNA Polymerase-Promoter Binding, Isomerization to Initiation-Competent Open Complexes, and Initiation of RNA Synthesis. J Mol Biol. 2011;412:754–71. - PMC - PubMed
    1. Gries TJ, Kontur WS, Capp MW, Saecker RM, Record MT., Jr. One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex. Proc Natl Acad Sci USA. 2010;107:10418–23. - PMC - PubMed
    1. Kontur WS, Capp MW, Gries TJ, Saecker RM, Record MT., Jr. Probing DNA Binding, DNA Opening, and Assembly of a Downstream Clamp/Jaw in Escherichia coliRNA Polymerase-λPR Promoter Complexes Using Salt and the Physiological Anion Glutamate. Biochemistry. 2010;49:4361–73. - PMC - PubMed
    1. Kontur WS, Saecker RM, Capp MW, Record MT., Jr. Late steps in the formation of the E. coli RNA polymerase-λPR promoter open complexes: Characterization of conformational changes by rapid [perturbant] upshift experiments. J Mol Biol. 2008;376:1034–47. - PMC - PubMed

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