Post-transcriptional regulation of RpoS synthesis

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EXCEED THE SPACE PROVIDED. RpoS is a sigma factor, discovered and best studied in the enteric bacteria, that is important in orchestrating responses to many stresses. RpoS activity is greatly increased during stationary phase after growth in rich medium, by limitation for individual nutrients (e.g. carbon or nitrogen), by high osmolarity medium, and after entry into the eukaryotic host cell, among other stimuli. Expression of more than 50 genes responds to RpoS, and the cognate gene products act to mitigate the adverse consequences of stress for the cell. RpoS matters in the real world, where "feast and famine" is the norm. Our goal is to understand the mechanisms regulating RpoS abundance, which are poorly understood. The principal control occurs by post-transcriptional regulation of RpoS synthesis, and by regulated protein turnover. We focus here on the control of RpoS synthesis. Escherichia coil is our model organism, but the results should be broadly applicable, in two senses. First, they should illuminate the important role of RpoS in pathogenic genera such as Salmonella and Yersinia, and they will also advance our understanding of post-transcriptional gene regulation. Genetic analysis has suggested that one known RNA-binding protein, Hfq, and another possible RNA- binding protein, DksA, are likely to interact with rpoS mRNA to control its expression. The small molecule "alarmone" ppGpp also has a role. The target mRNA has an antisense element that pairs with the ribosome binding site to limit translation. The function of the antisense element is counteracted in a way that requires the RNA-binding proteins and under at least some conditions, a trans-acting anti-antisense RNA. Experiments described in the specific aims utilize mainly genetic but also physical approaches: to verify the secondary structure of the rpoS mRNA, to identify the important proximal factors and their sites of action, and to determine exactly what happens to this mRNA to increase its expression. PERFORMANCE SITE ========================================Section End===========================================