Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of ProQ.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and noncoding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in , 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 ProQ's FinO domain and explore its mechanism of RNA interactions.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of ProQ.

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 , though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of and binding assays to confirm key structural features of ProQ's FinO domain and explore its mechanism of RNA interactions.

A bacterial three-hybrid assay for forward and reverse genetic analysis of RNA-protein interactions.

This protocol describes a bacterial three-hybrid (B3H) assay, an in vivo system that reports on RNA-protein interactions and can be implemented in both forward and reverse genetic experiments. The B3H assay connects the strength of an RNA-protein interaction inside of living Escherichia coli cells to the transcription of a reporter gene (here, lacZ). We present protocols to (1) insert RNA and protein sequences into appropriate vectors for B3H experiments, (2) detect putative RNA-protein interactions with both qualitative and quantitative readouts and (3) carry out forward genetic mutagenesis screens.

Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter protein.

Noncoding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain sRNAs with proteins Hfq and ProQ.

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein.

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators.

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ.

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.