Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme.
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.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides; D011485:Protein Binding; D011487:Protein Conformation; D011721:Pyrenes; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D046911:Macromolecular Substances; D008301:Maleimides; D011721:Pyrenes; D012321:DNA-Directed RNA Polymerases