Probing the Molecular Mechanism of ProQ‐RNA Interactions using a Bacterial Three‐hybrid Assay

Abstract

In bacteria, small RNAs (sRNAs) are known to play important roles in gene regulation. The interactions between sRNAs and mRNAs are often facilitated by protein chaperones. Recently, ProQ has been shown to bind to dozens of E. coli sRNAs and mRNAs, and to facilitate base pairing between the mRNA hupA and sRNA RaiZ with the effect of regulating hupA. These observations have led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression. Our goal has been to understand the molecular mechanisms of ProQ’s interaction with RNAs, mapping the amino acids on ProQ’s surface and nucleotides of RNAs that contribute to binding and regulation. To do this, we have used a bacterial three‐hybrid (B3H) assay to detect ProQ‐RNA interactions genetically. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing an sRNA or mRNA of interest is tethered to a DNA sequence upstream of a test promoter. Interaction of ProQ with the RNA stabilizes the binding of RNAP to the test promoter and activates transcription of a reporter gene, lacZ. Using domain truncations, site‐directed mutagenesis, and an unbiased forward genetic screen, we have identified a surface on ProQ’s NTD that we believe to be the primary surface for recognition of these SibB and cspE RNAs in vivo, and have created a genetically guided model for ProQ binding to an RNA duplex. In addition, we are working to probe whether ProQ possesses unique surfaces that contribute to binding of certain sRNAs and/or mRNAs and to determine what aspects of RNA sequence and structure contribute to recognition by ProQ.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.