Conformational transition of Escherichia coli RNA polymerase induced by the interaction of σ subunit with core enzyme

Abstract

The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) 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-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma 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-sigma 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 sigma 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 less than or equal to 3X10(-10)M was estimated for the PM-sigma 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 degrees 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 sigma subunit with core enzyme presented here further supports the proposition that the sigma 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.

Interaction of σ70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance

“…The binding and kinetic parameters of the c N^c ore polymerase interaction are similar to those previously established for the c 70^c ore interaction using an Npyrene maleimide derivative of the c 70 factor as a £uorescent label to follow binding to core [10].…”

“…This is the case in binding of c to core, where initial rapid bimolecular association is followed by slower conformational changes [9,10]; the association process will be kinetically biphasic, and may well be a¡ected by mass transport [11]. Evaluation of the dissociation phase is usually simpler, and essentially represents exponential dissociation of the complex limited by the slowest step.…”

“…1 all ¢tted well (M 2 = 0.02, R = 0.95) to a single exponential decay function, and on the basis that this represents a single dissociation process, the mean dissociation rate constant was evaluated to be 4.0 ( þ 1.0)U10 33 s 31 . Although the kinetics of c^core interactions have been studied for c 70 [10] and c N [9] and it has been demonstrated that in both cases c induces conformational changes in the resulting holoenzymes, little direct information exists on the c release step, which has been assumed to be relatively slow. The kinetic results presented here support this assumption, and suggest that, in the absence of DNA or transcription, the half-life of the c 70^c ore complex is about 200 s. Our results are in broad agreement with those of Fenton et al [12], who observed half-lives in the range 4^11 min for wild-type and a range of mutant c 70 factors.…”

The Bacteriophage T4 Inhibitor and Coactivator AsiA InhibitsEscherichia coliRNA Polymerase More Rapidly in the Absence of σ70Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70and Core

“…needed for the dynamic equilibrium between holoenzyme and 70 plus core to make free 70 available for binding to AsiA (6,12,14,24,37,41). Thus, two simple models could explain the ability of AsiA to inhibit E ⌬1.1 more readily than E fl .…”

“…These results suggest that the presence of 70 region 1.1 substantially increases the stabilization of the 70 /core interaction. Previous work has determined that the interaction of 70 with core is quite strong, with measured binding constants ranging from 2 ϫ 10 Ϫ7 to 5 ϫ 10 Ϫ10 M (12,14,24,41). While the primary interaction of 70 with core involves the interaction of 70 regions 2.1 to 2.2 with the coiled-coil region of ␤Ј (1), the overall interface of 70 with core is extensive, involving several regions of 70 and ␤/␤Ј (27,28,35,38).…”