Kinetic studies and structural models of the association of E. coli σ70 RNA polymerase with the λPR promoter: Large scale conformational changes in forming the kinetically significant intermediates

Ruth M. Saecker, Oleg V. Tsodikov, Kristi L. McQuade, Peter E. Schlax, Michael W. Capp, M. Thomas Record

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96 Scopus citations

Abstract

The kinetics of interaction of Eσ70 RNA polymerase (R) with the λPR promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42°C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I2, RPo) are single-exponential with first order rate constant βCR. Interpretation of the polymerase concentration dependence of βCR in terms of the three step mechanism of open complex formation yields the equilibrium constant Κ1 for formation of the first kinetically significant intermediate (I1) and the forward rate constant (κ2) for the conformational change converting I1 to the second kinetically significant intermediate I2:R + P ⇄Κ1 I1k2 I2. Use of rapid quench mixing allows Κ1 and κ2 to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (> 60 bp) interface formed in I1, its relatively small binding constant Κ1 at 37°C at this [salt] (∼ 6 × 106 M-1) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of κa (ΔCoa/o,‡ = -1.5(±0.2) kcal Κ-1), now shown directly to originate largely a from formation of I1 (ΔC1/o = -1.4(±0.3) kcal Κ-1), rather than from the formation of I2 as previously proposed. The isomerization I1 → I2 exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy (E2/act = 34(±2) kcal). This kinetic signature suggests that formation of the transition state (I1-I2)‡ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I1 involves coupled folding of unstructured regions of polymerase (β, β′ and σ70) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of σ70 and possibly domain 1 of β induce a kink at the -11/-12 base pairs of the λPR promoter which places the downstream DNA (-5 to +20) in the jaws of the β and β′ subunits of polymerase in I1. These early interactions of β and β′ with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.

Original languageEnglish
Pages (from-to)649-671
Number of pages23
JournalJournal of Molecular Biology
Volume319
Issue number3
DOIs
StatePublished - 2002

Bibliographical note

Funding Information:
We thank B. Ason for assistance with preliminary KinTek experiments, C. Farmer and W. Kontur for preparing and purifying unlabeled and 3 H-labeled λP R DNA fragments and R. Spolar for valuable laboratory assistance. We acknowledge helpful discussions with K. Adelman, M. Barker, C. Bingman, M. Buckle, R. Burgess, E. Courtenay, M. Craig, S. Darst, R. Gourse, C. Gross, R. Landick, M. Levandoski, M. Raffaelle, P. Schlax and W. Ross during the course of this work. We thank M. Buckle, P. deHaseth, E.P. Geidushek, G. Gussin, T. Heyduk, A. Hochschild, W. Ross, B. Sclavi, and P.H. von Hippel for constructive comments on the manuscript. We are grateful to I. Artsimovitch for discussions, comments and sharing Ref. 49 in advance of publication, and to S. Darst for sharing Refs. 57,58 in advance of publication. This research was supported by NIH grant GM23467.

Keywords

  • Mechanism
  • Protein folding
  • RNA polymerase
  • Transcription initiation

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology

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