Abstract 1964 Investigating the RNA-binding mechanism of bacterial KH-domain proteins

RNA-binding proteins serve as important modulators of RNA stability and interactions, and often support the function of regulatory small RNAs (sRNAs). While many bacteria possess global RNA-binding proteins such as ProQ and Hfq, these proteins are missing from many bacterial species including C. jejuni and H. pylori. Two KH-domain proteins, KhpA and KhpB, have recently been found to act as global RNA-binding proteins regulating gene expression in several bacterial species. However, the RNA-binding mechanisms of these KhpA/B proteins have not been well established. Given that the KH-domains of these proteins are able to dimerize, it is also possible that there are distinct RNA-binding activities for homo- and hetero-dimeric complexes. This project therefore aims to dissect the molecular interactions of KhpA and KhpB with each other and with RNA. To do so, we are using two genetic techniques - a bacterial-two-hybrid (B2H) and a bacterial-three-hybrid (B3H) assay - that allow for in vivo detection of protein-protein and RNA-protein interactions, respectively. The B2H assay is able to detect heterodimerization of KhpA/B proteins from several species, as well as homodimerization of a subset of these proteins. Preliminary B3H results suggest that individual KhpA/B proteins vary widely in their strength and specificity of RNA binding, and we are using a combination of forward and reverse genetics to identify the RNA-binding surfaces of these proteins. Ongoing work aims to establish a B3H system that can assess RNA binding in the context of a heterodimeric KhpA/B pair, which may be the most functionally relevant configuration of these proteins. This project should uncover mechanistic insights into how bacterial KH-domain proteins interact with each other and with their RNA ligands in order to regulate gene expression. We gratefully acknowledge funding support from NIH (R15GM135878), the Camille and Henry Dreyfus Foundation and Mount Holyoke College.