Elucidation of the mechanisms of RNA binding by the Escherichia coli ProQ N-terminal domain: a molecular genetic approach
Date
2021-05-26
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
ProQ is a global RNA-binding protein that is present in many bacterial organisms and helps control gene expression by binding to numerous RNA partners including sRNAs and mRNA UTRs. Although Escherichia coli ProQ’s structure has been solved with NMR spectroscopy, there is still no co-structure of ProQ bound to its RNA partners, so we do not have a full understanding of ProQ’s mechanism of interaction with RNA.
In order to probe this mechanism, we have used a bacterial three-hybrid assay with a genetic lacZ reporter. With this tool, we can use molecular genetics to test many mutant proteins in a relatively short amount of time. Here, I discuss a dissection of ProQ’s RNA-binding mechanisms focusing on its FinO-like domain which is the N-terminal domain (NTD) of Escherichia coli ProQ.
This study includes (1) the exploration of two surface-exposed aspartate residues on the concave face of ProQ’s NTD, as described in a previously published study. Three more recent projects focusing on (2) probing the contributions to RNA binding of potential hydrogen-bonding residues on the convex face of ProQ’s NTD; (3) using double substitutions to detect more subtle or redundant effects of residues; and (4) using an RNA substrate with an extended single stranded poly(uridine) tail to determine RNA-specificity of these effects. We demonstrate that the convex face of ProQ’s NTD does interact with RNA and that there may be some RNA-specific dependence on certain convex residues.
Description
Keywords
RNA-protein interactions, ProQ, Bacterial gene regulation, Escherichia Coli