Conformational Transition of Escherichia coli RNA Polymerase Induced by the Interaction of α Subunit with Core Enzyme

Felicia Y.H. Wu, Lynwood R. Yarbrough*, Cheng Wen Wu

*Corresponding author for this work

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

The isolated σ subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(l-pyrene)maleimide. The labeled σ subunit (PM-σ) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-σ. there was a decrease in fluorescence intensity without shifts in emission maxima of PM-σ. The kinetics of the interaction between the σ subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-σ was rapidly mixed with an excess of core enzyme. The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of ≤ 3 × 10-10 M was estimated for the PM-σ-core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 °C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the “overall” conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of subunit with σ core enzyme presented here further supports the proposition that the σ subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.

Original languageEnglish
Pages (from-to)3254-3258
Number of pages5
JournalBiochemistry
Volume15
Issue number15
DOIs
StatePublished - 1 Jul 1976

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