Analysis of the RNA-binding properties of Hfq proteins from the bacteria Escherichia coli, Listeria monocytogenes, and Caulobacter crescentus
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Bacterial small, noncoding (s)RNAs play important roles in post-transcriptional gene regulation through complementary base-pairing interactions with target messenger (m)RNA molecules,. Many sRNAs rely on protein partners to carry out their regulatory functions. As such, the interactions between proteins and small RNAs contribute to gene regulation across all three domains of life. In particular, the hexameric RNA chaperone protein, Hfq, has been shown to mediate interactions between bacterial small (s)RNAs and their target mRNAs as a part of bacterial stress responses. Hfq orthologues have been characterized in approximately half of all sequenced bacterial species. This protein functions to stabilize sRNA steady-state expression levels, protect sRNAs from cleavage, accelerate efficient base pairing between sRNA and mRNA to activate or repress translation, and may recruit cellular endonucleases to degrade the transcript following repression. To examine the RNA- and protein-binding properties of Hfq proteins in Escherichia coli, Listeria monocytogenes, and Caulobacter crescentus, I utilized a set of two transcription-based bacterial-hybrid assays designed to detect these interactions in vivo. In the bacterial two-hybrid assay (B2H), Hfq monomers from each bacterial species displayed inter- and intra-species multimerization, supporting the notion that hexamerization is a conserved feature of Hfq orthologs. Through the utilization of a novel bacterial three-hybrid assay designed to detect RNA-protein interactions in vivo, I report that the Hfq protein from each of these species is able to recognize a variety of sRNAs with unique binding patterns. A comparative analysis of the relative interaction strengths of Hfq from each of these species with a panel of E. coli sRNAs shows that the C. crescentus Hfq often displays stronger sRNA-binding interactions than E. coli Hfq and L. monocytogenes Hfq. Further analysis of these three proteins at the level of primary sequence and tertiary structure suggests that the presence of certain ionizable amino acid residues in the Hfq sequence may play particularly important roles in binding sRNAs across bacterial species.