Probing the molecular mechanism of ProQ‐RNA interactions using a bacterial three‐hybrid assay

Abstract

In bacteria, small RNAs (sRNAs) play important roles in gene regulation; sRNAs can regulate the translation and stability of target mRNAs via imperfect base pairing. These sRNA‐mRNA interactions are often facilitated by RNA chaperone proteins. ProQ has recently been identified as a global RNA‐binding protein that binds to dozens of sRNAs and hundreds of mRNAs in multiple proteobacteria; this observation has led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression.Our goal is to understand the molecular mechanisms of ProQ's interaction with regulatory RNAs, mapping the amino acids on ProQ's surface and nucleotides of RNAs that contribute to binding and regulation by using a bacterial three‐hybrid (B3H) assay to genetically detect ProQ‐RNA interactions. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing either an sRNA or 3′UTR 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.We have detected preliminary B3H interactions of ProQ with several of its RNA partners. Our data supports a model where the conserved N‐terminal‐domain (NTD) is the primary region for RNA binding. Further, we have identified point mutations in ProQ that affect its RNA interactions and have used immunodetection to determine ProQ expression levels to ensure that these mutations are not destabilizing. Current efforts are focused on screening for additional ProQ point mutations to locate the binding interface(s) for the sRNAs and mRNAs with which it interacts as well as exploring the structure and sequences of RNA that are required for ProQ interaction.Support or Funding InformationResearch supported by Mount Holyoke College and the Luce FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.