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.
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.
Non‐coding small regulatory RNAs (sRNAs) have an important role in bacterial stress responses. The binding of sRNAs to their target mRNAs is facilitated by RNA‐binding proteins such as Hfq or ProQ. The Berry Lab has developed a bacterial three‐hybrid (B3H) assay to detect the binding of RNA with both of these RNA chaperones in vivo, by connecting the strength of an RNA‐protein interaction to the expression of a reporter gene. The interaction between a “prey” protein fused to the α‐subunit of RNA polymerase (RNAP) and a “bait” RNA tethered upstream of a test promoter stabilizes the binding of RNAP and increases transcription of the reporter gene lacZ. Despite the promise of the B3H system and its success in detecting many high‐affinity interactions, low signal‐to‐noise for other RNA‐protein interactions currently limits the broader utility of the assay. Computational structure predictions suggested that certain RNAs of interest could misfold when hybridized with other components of the hybrid RNA construct, e.g. an MS2 hairpin (MS2hp) or an exogenous intrinsic terminator. Such misfolding would likely disrupt a bait RNA’s interaction with the prey protein. To avoid this limitation, this study aims to optimize the hybrid RNA construct to improve the breadth of detectable interactions in the B3H assay. To this end, we designed new hybrid RNA constructs with the addition of a GC‐clamp – a short insert of guanines (G) and cytosines (C) flanking a region of interest – to promote proper folding and optimal display of RNA. Several sRNAs and 5’UTRs were cloned into GC‐clamp designs and their in vivo interactions with Hfq were tested in the B3H assay. Preliminary results demonstrate the promise of a short GC‐clamp in improving the B3H signal for many sRNA‐Hfq interactions, and we are currently working to test these GC constructs with additional RNAs. Increasing the sensitivity and generalizability of the B3H assay to study bacterial RNA‐protein interactions will help shed light on the molecular mechanisms of RNA‐chaperone proteins and the important processes in bacteria they regulate, such as adaptation to stress, biofilm formation and virulence.
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