The RpoN regulon of Salmonella and its component RNA repair system

Abstract

RNA polymerase (RNAP) holoenzyme catalyzes transcription in eubacteria. The one variable subunit of this multi-subunit enzyme is σ, which mediates promoter recognition and promoter-holoenzyme interactions. In addition to a primary σ-factor, most cells have one or more alternative σ-factors that regulate different sets of genes in response to varying environmental conditions. One alternative σ-factor, σ54, differs from other known σ-factors in structure, conserved promoter elements, and in its absolute requirement for an activator protein that, upon stimulation by a particular environmental condition, interacts with RNAP-σ54 and hydrolyzes ATP, generating the energy necessary for transcriptional initiation. This activator requirement can hinder global analysis of the σ54 regulon because σ54-dependent promoters will be transcriptionally silent without proper environmental cues. To overcome this limitation, an engineered promiscuous, constitutively-active variant of the Sinorhizobium meliloti DctD activator was used to define the σ54 regulon in Salmonella Typhimurium. Using this engineered activator, microarray analysis was used to identify σ54-dependent transcripts. This approach confirmed the regulation of 16 promoters previously predicted to be σ54-dependent. Chromatin immunoprecipitation linked to microarray analysis revealed 70 sites throughout the genome interacting with either σ54 or RNAP-σ54. Surprisingly, >50% of these sites were predicted to fall within coding sequences. Promoter fusion assays indicated that some of these intragenic sequences could function as σ54-dependent promoters , raising the possibility of new regulatory roles for σ54. One operon that was shown to be σ54-dependent encodes a putative RNA repair system. The components of this system—an RNA-binding ribonucleoprotein complex (Rsr-Y RNA), an RNA ligase (RtcB), and an RNA phosphate cyclase (RtcA)—are found throughout all domains of life. While functions for eukaryotic/archaeal homologs have been described, their role in bacteria remains enigmatic, largely because conditions that stimulate the activator protein, allowing transcription are unknown. We used quantitative, reverse transcriptase PCR to assess transcription after exposure to various stresses. Treatment with Mitomycin C, a nucleic acid alkylating agent, resulted in up-regulation of this operon. This finding supports our model in which these genes are expressed in response to nucleic acid damage, and their products interact with the damaged molecules to directing their repair or degradation.