Small RNAs (sRNAs) have been propelled to the forefront of genetic research as their diverse roles in regulation of cellular processes have been recognized. sRNAs are crucial for development in higher organisms; in bacteria, sRNAs regulate responses to many environmental stresses. Bacterial sRNAs that require the RNA chaperone Hfq regulate the translation or stability of mRNA targets by base pairing-dependent mechanisms. The Hfq-dependent sRNA SgrS is essential in E. coli for the response to glucose- phosphate stress. SgrS represents a new paradigm for sRNA regulators in bacteria. Other characterized Hfq-dependent sRNAs are non-coding and function solely via base pairing with and regulating target mRNAs (riboregulation). SgrS performs riboregulation on mRNA targets, and additionally encodes a novel protein, SgrT. This proposal focuses on the riboregulation function of SgrS, which our preliminary data indicates is more evolutionarily conserved than the protein coding function. Preliminary microarray experiments identified 10 candidate SgrS target mRNAs and we provide further evidence that 3 of these are directly up- or down-regulated by base pairing with SgrS. Analysis of SgrS homologs and their putative targets suggests that SgrS:mRNA interactions are better conserved for some targets than others. Since regulation of multiple targets by a single sRNA is a common theme for both bacterial and eukaryotic sRNA regulators, we propose to use SgrS as a model to examine how regulation of multiple targets with different base pairing characteristics and affinities is coordinated. Experiments in Aim 1, will define the SgrS target regulon. In Aim 2, a high- throughput RNA footprinting approach will be used to map sRNA:mRNA interactions for all E. coli SgrS:mRNA targets and some homologous SgrS:mRNA pairs with predicted unique base pairing characteristics. These studies will demonstrate direct interactions between SgrS and its targets and define the molecular determinants of these interactions. In Aim 3, we will determine whether there is a mechanism for SgrS to prioritize regulation of targets when SgrS levels are limiting (i.e., establish a hierarchy). We will then alter base pairing interactions by mutagenesis and determine how these alterations affect the regulatory hierarchy.