Genetic Identification of Bacterial RNA Chaperone Proteins and their Mechanisms of Action

Project Summary
Regulatory small RNAs (sRNAs) play important roles in stress responses in nearly all bacterial
organisms that have been investigated, including pathogenic bacteria. The functions of sRNAs
are often supported by proteins such as the paradigmatic bacterial RNA chaperone protein, Hfq.
Interestingly, only ~50% of bacteria possess a Hfq protein, despite many of these organisms,
including important human pathogens, being known to utilize sRNAs. A less well characterized
RNA chaperone called ProQ is present in a subset of Hfq-containing organisms and, like Hfq,
binds dozens of sRNAs and messenger (m)RNAs with regulatory consequences. In addition, a
pair of KH-domain proteins—KhpA and KhpB—have recently been found to act as global RNA-
binding proteins (RBPs) in several bacterial species. Their phylogenetic distribution raises the
intriguing possibility that KhpA or B proteins—or a heterodimeric complex of the two—may
support the function of sRNAs in bacteria without Hfq or ProQ proteins. While much is known
about the mechanism of Hfq-RNA interactions, significant gaps remain in our understanding of
how other bacterial RNA chaperones (e.g ProQ, KhpA and KhpB) interact with RNA to regulate
gene expression. This proposal builds on an innovative genetic approach to probe RNA-protein
interactions inside of E. coli cells with a transcription-based bacterial three-hybrid (B3H) assay.
This assay can detect interactions in the native context of a bacterial cytoplasm and offers a
straightforward genetic approach to both identify and assess the consequences of mutations in
RBPs with molecular phenotypes of interest. The long-term goal of the PI’s laboratory is to
understand the molecular mechanisms by which RNA chaperones interact with sRNAs and
mRNAs to drive bacterial gene regulation. This proposal aims to extend the capabilities of the
B3H assay to address current limitations of the system. Aim 1 will adapt constructs to support co-
expression of multiple heterologous proteins that may collaborate in RNA interaction. Aim 2 will
develop versatile options to precisely control how bait RNAs are synthesized and presented in
the cell. As part of both aims, newly developed constructs will allow important biological questions
to be addressed—from characterizing KH-domain proteins from multiple bacterial species to
dissecting ProQ’s interactions with mRNAs. The proposed research is innovative because it
approaches the analysis of bacterial RBPs through the development and application of a unique
genetic methodology with the potential to be a useful tool for many in the field. The project is
significant as knowledge gained about mechanisms of bacterial RNA-protein interactions will
increase the potential of RNA chaperones to serve as therapeutic targets for bacterial infections.

Grant Number: 2R15GM135878-02
NIH Institute/Center: NIH
Principal Investigator: Katherine Berry