Identifying species‐specific Hfq‐RNA Interactions using a bacterial three‐hybrid assay

Abstract

Bacteria use non‐coding small RNAs (sRNAs) to regulate expression of target mRNAs, often with the help of protein chaperones. About half of bacterial species possess a well‐characterized globally‐acting RNA‐binding protein, a hexameric protein called Hfq, which contributes to virulence in many pathogenic species. While much is known about the mechanism of RNA interactions by Hfq proteins in certain well‐studied model bacteria, there are significant gaps in our understanding of how conserved the RNA‐binding behavior of Hfq homologs from diverse bacterial organisms. Here, we use a recently developed bacterial three‐hybrid (B3H) assay to further define the interactions between Hfq and its RNA substrates. This assay connects the interaction between a DNA‐bound RNA and a RNA polymerase (RNAP)‐bound protein Hfq to the expression of a reporter gene, providing a genetic route to the analysis of RNA interactions from a heterologously expressed RNA‐binding protein. This provides a facile route to study the behavior of diverse RNA‐binding proteins in an innocuous laboratory strain of E. coli. Here we present data collected using the B3H assay to compare the RNA interactions of Hfq from several bacterial species, including Listeria monocytogenes, Caulobacter crescentus, and Escherichia coli. We have observed distinct RNA‐binding profiles between these Hfq proteins. Sequence alignments of these three proteins led to a hypothesis that charged residues along the rim surface of Hfq may underlie the differences in binding preferences for different sRNAs. Data will be presented that test this model using both chimeric and site‐directed mutants. Our long‐term goal is to gain a better understanding of what structural elements of an RNA‐binding protein such as Hfq determine its RNA binding and function in driving bacterial gene regulation.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.