Abstract:Transcription is the first and most heavily regulated step in gene expression. Sigma () factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the 70 family of proteins and the essential roles they play in the transcription process.Key words: transcription, bacteria, RNA polymerase, sigma factor, gene expression.
σ factors are single subunit general transcription factors that reversibly bind core RNA polymerase and mediate gene-specific transcription in bacteria. Previously, an atypical two-subunit σ factor was identified that activates transcription from a group of related promoters in Bacillus subtilis. Both of the subunits, named SigO and RsoA, share primary sequence similarity with the canonical σ70 family of σ factors and interact with each other and with RNA polymerase subunits. Here we show that the σ70 region 2.3-like segment of RsoA is unexpectedly sufficient for interaction with the amino-terminus of SigO and the β' subunit. A mutational analysis of RsoA identified aromatic residues conserved amongst all RsoA homologues, and often amongst canonical σ factors, that are particularly important for the SigO-RsoA interaction. In a canonical σ factor, region 2.3 amino acids bind non-template strand DNA, trapping the promoter in a single-stranded state required for initiation of transcription. Accordingly, we speculate that RsoA region 2.3 protein-binding activity likely arose from a motif that, at least in its ancestral protein, participated in DNA-binding interactions.
Pseudomonas aeruginosa is a ubiquitous environmental bacterium, which has emerged as an opportunistic pathogen of major clinical relevance (Bassetti et al., 2018). Its extensive metabolic versatility facilitates survival not only in different aquatic and terrestrial habitats, but also in the human host. P. aeruginosa infections can become established at various host sites and progress into life threatening acute or chronic infections (Lorenz et al., 2019;Moradali et al., 2017;Turner et al., 2014). The pathogen is equipped with a large arsenal of virulence factors. Expression of many of the respective P. aeruginosa virulence genes is orchestrated by two hierarchical organized homoserine lactone (HSL)-dependent quorum-sensing (QS) systems, the las and the rhl system (
Sigma (σ) factors are single-subunit proteins that reversibly bind RNA polymerase and play an important role in the transcription initiation process. An unusual 2-subunit σ factor, consisting of proteins SigO and RsoA, activates transcription from a group of related promoters in Bacillus subtilis. These 2 proteins specifically interact with each other and with RNA polymerase subunits. This system is widespread among species in several Bacillus-related genera, but otherwise appears restricted to the Firmicutes. Here, we reconstituted SigO-RsoA, and a cognate promoter, into the distantly related heterologous host Escherichia coli to examine whether this system can function in bacteria outside of the Firmicutes. We show that these proteins can productively associate with E. coli RNA polymerase and activate transcription, demonstrating that there are no structural barriers to function. In parallel, we tested a wide array of protein-protein interaction mutations and promoter mutations that impact SigO-RsoA function in both B. subtilis and E. coli and conclude that the SigO-RsoA system behaves, in most instances, similarly in both genetic backgrounds. These data raise the possibility of genetically isolating the system in this heterologous host and away from unknown B. subtilis factors that may also be playing a role in SigO-RsoA regulatory pathways, thus facilitating further study of the system. As a result of this work, we also provide a comprehensive mutational analysis of a SigO-RsoA promoter and report the preliminary identification of amino acids in SigO that play a role in mediating the SigO-RsoA protein-protein interaction.
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