Project Summary Regulatory small RNAs (sRNAs) play important roles in stress responses in nearly all bacterial organisms that have been investigated, including pathogenic bacteria. The functions of sRNAs are often supported by proteins such as the paradigmatic bacterial RNA chaperone protein, Hfq. Interestingly, only ~50% of bacteria possess a Hfq protein, despite many of these organisms, including important human pathogens, being known to utilize sRNAs. A less well characterized RNA chaperone called ProQ is present in a subset of Hfq-containing organisms and, like Hfq, binds dozens of sRNAs and messenger (m)RNAs with regulatory consequences. In addition, a pair of KH-domain proteins—KhpA and KhpB—have recently been found to act as global RNA- binding proteins (RBPs) in several bacterial species. Their phylogenetic distribution raises the intriguing possibility that KhpA or B proteins—or a heterodimeric complex of the two—may support the function of sRNAs in bacteria without Hfq or ProQ proteins. While much is known about the mechanism of Hfq-RNA interactions, significant gaps remain in our understanding of how other bacterial RNA chaperones (e.g ProQ, KhpA and KhpB) interact with RNA to regulate gene expression. This proposal builds on an innovative genetic approach to probe RNA-protein interactions inside of E. coli cells with a transcription-based bacterial three-hybrid (B3H) assay. This assay can detect interactions in the native context of a bacterial cytoplasm and offers a straightforward genetic approach to both identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. The long-term goal of the PI’s laboratory is to understand the molecular mechanisms by which RNA chaperones interact with sRNAs and mRNAs to drive bacterial gene regulation. This proposal aims to extend the capabilities of the B3H assay to address current limitations of the system. Aim 1 will adapt constructs to support co- expression of multiple heterologous proteins that may collaborate in RNA interaction. Aim 2 will develop versatile options to precisely control how bait RNAs are synthesized and presented in the cell. As part of both aims, newly developed constructs will allow important biological questions to be addressed—from characterizing KH-domain proteins from multiple bacterial species to dissecting ProQ’s interactions with mRNAs. The proposed research is innovative because it approaches the analysis of bacterial RBPs through the development and application of a unique genetic methodology with the potential to be a useful tool for many in the field. The project is significant as knowledge gained about mechanisms of bacterial RNA-protein interactions will increase the potential of RNA chaperones to serve as therapeutic targets for bacterial infections.