The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5′ and 3′ untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ’s interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ’s NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs.Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex. INTRODUCTION Regulatory, small RNAs (sRNAs) are found in nearly all bacterial species and implicated in important processes such as virulence, biofilm formation, host interactions and antibiotic resistance.(1-3) These sRNAs typically regulate messenger RNA (mRNA) translation through imperfect base pairing near an mRNA's ribosomal binding site.(2, 4-6) In many bacterial species, the stability and function of sRNAs are supported by global RNA-binding proteins, such as the protein Hfq.(1, 4, 7-9) Given that Hfq is not present in all bacterial species and that not all sRNAs depend on Hfq for their function, there is increasing interest in other RNA-binding proteins that may play a role in global gene-regulation in bacteria,(2, 10-13) including a class of proteins that contain FinO domains.(14-17) The Escherichia coli protein FinO is the founding member of the FinO structural class of RNA-binding proteins. In E. coli, FinO binds the FinP sRNA and regulates the 5´ untranslated region (UTR) of traJ.(18, 19) Similarly, Legionella pneumophila RocC contains a FinO-domain and binds the sRNA RocR along with at least four 5' UTRs of mRNAs involved in competence.(20) In E. coli, another FinO-domain-containing protein called ProQ was initially characterized as an RNA-binding protein contributing to osmoregulation through expression of proP.(21) ProQ was recently identified through Grad-Seq experiments to bind to dozens of cellular RNAs,(17) including a large number of sRNAs and mRNA 3'UTRs in Samonella and E. coli.(22) ProQ binding has been shown to regulate mRNA-expression levels through interactions with both 5' and 3' UTRs. It has been shown to form a ternary complex with an sRNA (RaiZ) and an mRNA (hupA), to support RaiZ's repression of hupA,(23) and to protect mRNAs from exonucleolytic degradation by binding to 3' ends.(22) Further, ProQ supports the sRNA SraL in preventin...

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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.