Generation of single-cysteine E. coli ProQ variants to study RNA-protein interaction mechanisms

Abstract

ProQ is a FinO-domain protein found in E. coli and other proteobacteria that has a global RNA-binding profile. In order to probe the detailed mechanism of RNA interactions, we have developed a collection of 13 E. coli ProQ variants that possess single-cysteine residues at varied positions on the surface of the N-terminal FinO domain and retain the ability to bind well to RNA. This set of variant ProQ proteins will support future biochemical and biophysical studies to map the orientation of bound RNAs to different sites around the ProQ protein, shedding light on the mechanism of ProQ-RNA interactions.

Figure 1. Testing the RNA-binding ability of E. coli ProQ single-cysteine variants

a) Domain- and b) protein-structure of E. coli ProQ, with the position of the two native cysteine residues highlighted. The 3D protein structure was predicted by AlphaFold (Jumper et al. 2021; Varadi et al. 2022). c) Schematic of bacterial three-hybrid (B3H) system to detect interaction between ProQ ^NTD and malM 3ʹUTR RNA, fused to the NTD of the α subunit of RNAP and an MS2 RNA hairpin (MS2 ^hp ; see methods), respectively. d-f) Results of β-galactosidase assays testing the interaction between ProQ ^NTD and malM- 3’UTR using the B3H assay (Stockert et al. 2022; Pandey et al. 2020). The interactions of ProQ ^NTD mutants lacking one or both native cysteine residues are compared to wild-type (WT) ProQ. Data are plotted with varying levels of normalization: d) raw β-galactosidase data are shown for each transformation of KB473 reporter cells with pPrey-ProQ ^NTD (pKB951 or a mutant derivative) and pBait- malM -3’ (pKB1210) or the corresponding negative controls (alpha empty and pCH1, respectively). The fold-stimulation over basal levels of lacZ activity is defined as the ratio of lacZ activity when both bait and prey are present divided by the highest value from the negative controls when either bait or prey is missing. e) B3H data of additional single-cysteine ProQ ^NTD mutants are plotted as fold-stimulation over basal levels of lacZ activity. f) B3H data of single and double-cysteine ProQ ^NTD mutants are plotted as normalized fold-stimulation values, where the fold-stimulation of ProQ ^WT is set to 1.0 and a fold-stimulation of 1.0, representing no B3H interaction, is set to 0 (see Methods). g) B3H data comparing interaction of ProQ ^C24S-C88A to versions of this cysteine-free ProQ ^NTD containing additional mutations introducing single cysteine residues. Data are plotted as normalized fold-stimulation values, where the fold-stimulation of ProQ ^C24S-C88A is set to 1.0. Data from mutants producing at least 70% of the interaction of ProQ ^C24S-C88A are shown in magenta and those <70% are shown in yellow. In c-f), bar graphs show the average and standard deviations of values collected from three or more independent experiments conducted across multiple days. h) Locations of residues substituted with single-cysteine mutations are shown on the AlphaFold-predicted structure (Jumper et al. 2021; Varadi et al. 2022); residues are colored as in (g) based on the strength of RNA-binding of the ProQ ^NTD variant with a cysteine at each position.