Characterization of structurally atypical sigma factors carrying fused domains of unknown function in the plant endosymbiont, Sinorhizobium meliloti

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Transcription is the first major step in gene expression in all organisms. In bacteria, all gene expression is regulated by a family of general transcription factors called sigma () factors. Each factor controls the expression of a subset (its regulon) of genes and regulons may consist of between one and thousands of genes depending on the factor. An important group of these proteins is comprised of Extracytoplasmic Function (ECF) factors and the activity of many of these is negatively regulated by a signal transduction pathway that includes an antagonistic anti- factor protein. ECF factors and their associated regulators constitute a major sensory system that, along with specific transcription factors and two-component regulatory systems, mediate the cellular ability to mount appropriate gene expression responses to changing physicochemical conditions inside and outside of the cell. Mechanistically, factors carry out two major functions: they target RNAP holoenzyme to the gene promoter and they trap the transcription start site in a single-stranded state called an open-complex. Stabilization of the open-complex is an essential step in the transcription initiation pathway and factor activity is partially analogous to the activity of TFIIB in archaeal and eukaryotic RNAP II. Understanding mechanistic detail of factor function is a very active area of research that involves a few well-characterized model systems. However, the analysis of genome sequence data has revealed the presence of structurally unusual ECF factor-related proteins in many bacteria and these atypical or non-canonical systems remain understudied. Several sub-groups of ECF factors unexpectedly carry fused protein domains of unknown or incompletely characterized function. Emerging evidence points to regulatory functions for these fused domains and the long term objective of my research program is to study mechanistic details of non-canonical transcription systems in bacteria. The study of non-canonical systems carries the promise of uncovering novel pathways to transcription initiation and unexpected physiological relationships between the regulation of gene expression and other cellular processes. Remarkably, the important endosymbiotic bacterium Sinorhizobium meliloti synthesizes two unique examples of non-canonical fused domain factors. This current proposal describes the study of the physiological importance of one of these proteins (RpoE9) to S. meliloti biology. To these ends, we are proposing a series of research pursuits mainly focused on discerning unexpected intersections between RpoE9 activity and other cellular regulatory and homeostatic systems. We wish to emphasize that while the study of this structurally atypical factor is important in itself, we also view it as a vehicle to establish and refine the study of all ECF factors from a more global and systems-wide perspective.