Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ

Ewa M. Stein; Suxuan Wang; Katherine G. Dailey; Chandra M. Gravel; Shiying Wang; Mikołaj Olejniczak; Katherine E. Berry

doi:10.1261/rna.079697.123

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ

¹Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 61-614 Poznań, Poland
²Program in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts 01075, USA
³Department of Chemistry, Mount Holyoke College, South Hadley, Massachusetts 01075, USA

Corresponding authors: mol{at}amu.edu.pl, kberry{at}mtholyoke.edu

Abstract

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and noncoding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ’s FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA’s terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70, and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

Keywords

Received April 25, 2023.
Accepted August 3, 2023.

This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.