Evolving research on small RNAs (sRNAs) in bacteria implicates sRNAs as a key effector of gene regulation. While some sRNAs are able to act independently, many are dependent on an RNA‐binding protein, such as the well‐established Hfq in Escherichia coli. Another family of RNA‐binding proteins is the FinO family, including ProQ and FinO in E. coli, NMB1681 in N. meningitidis, and Lpp1663 in L. pneumophila. Structures for these proteins have been solved through both NMR and X‐ray diffraction, in addition to computational predictions. While many structural elements are common across all structures, there are interesting differences in regions that have been implicated by genetic experiments to be important for RNA binding. In order to investigate the structure and function relationships of these proteins, we have analyzed the available models for FinO family proteins to compare intriguing structural features, including the position and predicted contacts of a universally conserved arginine that plays a critical role in RNA binding. Finally, we are probing predicted interactions from structural models with the use of site‐directed mutagenesis and our laboratory’s bacterial three‐hybrid (B3H) assay. Together, this work is generating insights into the most relevant structural conformations for in vivo RNA binding by FinO proteins and the ways in which the structure of E. coli ProQ is both similar and distinct from orthologous FinO domain proteins.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ's FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA's terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70 and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.