Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofEscherichia coliProQ

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 inEscherichia 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 ofE. coliProQ’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 ofE. coliProQ, while additional contacts contribute to the binding of certain RNAs.

The FinO/ProQ-like protein PA2582 impacts antimicrobial resistance in Pseudomonas aeruginosa

“…As described for other ProQ homologs ( Olejniczak and Storz, 2017 ), the electrostatic surface potential of PA2582 shows that parts of α-helix 1 and the inner side of the U-shape protein region formed by the linker between α-helix 1 and the FinO domain, the FinO domain itself and the α-helix 6 are positively charged, which could suggest an involvement in RNA binding ( Supplementary Figure S1B ). In addition, the conserved residues Y70 and R80 ( E. coli numbering) that form the main RNA-binding site on this conserved concave face of ProQ Eco ( Stein et al, 2023 ) are present in PA2582 (corresponding to Y101 and R111 in PA2582). The third essential residue for RNA binding, R58 ( E. coli numbering) most likely corresponds to K89 in ProQ Pae , thus retaining the positive charge ( Stein et al, 2023 ; Supplementary Figure S1A ).…”

“…In addition, the conserved residues Y70 and R80 ( E. coli numbering) that form the main RNA-binding site on this conserved concave face of ProQ Eco ( Stein et al, 2023 ) are present in PA2582 (corresponding to Y101 and R111 in PA2582). The third essential residue for RNA binding, R58 ( E. coli numbering) most likely corresponds to K89 in ProQ Pae , thus retaining the positive charge ( Stein et al, 2023 ; Supplementary Figure S1A ). As PA2582 shows significant structural similarities to known ProQ homologs, we henceforward term this protein ProQ Pae .…”

“…Both domains are connected by an unstructured linker that is rich in positively charged amino acid (aa)-residues ( Gonzalez et al, 2017 ). While the NTD is the principal RNA binding site with a preference for highly structured RNAs containing double-stranded regions (e.g., intrinsic terminators), the CTD has a broader RNA binding specificity ( Pandey et al, 2020 ; Stein et al, 2020 , 2023 ).…”

ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis

“…Since the discovery of ProQ as a global RNA-binder in Salmonella and E. coli , a substantial amount of data regarding ProQ in vivo ligands, and ProQ-dependent gene expression, has become available ( 23 – 25 ). A deeper understanding about ProQ’s RNA-binding activity has emerged from biochemical, structural, and mutational studies ( 31 – 33 , 37 – 39 ). By contrast, mechanistic understanding linking ProQ binding events to effects on particular RNA ligands is limited to the study of a few examples.…”

Data_Sheet_1.pdf

“…Both domains are connected by an unstructured linker that is rich in positively charged amino acid (aa)-residues (Gonzalez et al, 2017). While the NTD is the principal RNA binding site with a preference for highly structured RNAs containing double-stranded regions (e.g., intrinsic terminators), the CTD has a broader RNA binding specificity (Pandey et al, 2020;Stein et al, 2020Stein et al, , 2023.…”