A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli

Abstract

Abstract 6S RNA is a bacterial transcriptional regulator, which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ70-containing RNA polymerase. This inhibitory RNA polymerase~6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs. Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6S RNA~pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem (conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.