Release of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA.

Abstract

To elucidate the mechanism of sigma release in the transcript by Escherichia coli DNA-dependent RNA polymerase, we obtained the time courses of sigma release and elongation of product RNA by a rapid kinetic technique; transcription was synchronously initiated from A1 promoter on T7 DNA by the addition of four substrates to a stoichiometric mixture of holoenzyme and template DNA, and then quenched by the addition of EDTA. The elongation rate was changed by limiting the concentration of one of four substrates, GTP. At reduced GTP concentration, elongation was decelerated, but the time course of sigma release was unchanged. No connection between sigma release and length of RNA product was found. The results lead to the conclusion that sigma is released depending only on time elapsed after initiation, not on the length of RNA product. We propose a two-step model for sigma release with a rapid triggering and a slow dissociation of about 5 s. This dissociation, the rate-determining step of sigma release, is independent of the rate of elongation.