Multiple Mechanisms of Transcription Inhibition by ppGpp at the λpR Promoter

Abstract

General stress conditions in bacterial cells cause a global cellular response called the stringent response. The first event in this control is production of large amounts of a regulatory nucleotide, guanosine-3',5'-(bis)pyrophospahte (ppGpp). It was proposed recently that ppGpp acts by decreasing stability of open complexes at promoters that make short-lived open complexes, e.g. the rRNA promoters. However, here we report that the bacteriophage lambdap(R) promoter, which forms long-lived open complexes, is inhibited by ppGpp in vitro as observed in vivo. We performed a systematic investigation of the ppGpp-specific inhibition of transcription initiation at lambdap(R) and found that ppGpp does decrease stability of open complexes at lambdap(R), but only slightly. Likewise the equilbrium binding constant and rate of open complex formation by RNA polymerase at lambdap(R) are only slightly affected by ppGpp. The major effect of ppGpp-mediated inhibition is to decrease the rate of promoter escape. We conclude that ppGpp-mediated inhibition of transcription initiation is not restricted to promoters that make short-lived open complexes. Rather we conclude that the initial catalytic step of transcript formation is affected by ppGpp, specifically formation of the first phosphodiester bond is inhibited by ppGpp at lambdap(R).