Structural dependence of ProQ-mRNA interactions in live bacterial cells

ProQ is a post-transcriptional gene regulator in bacteria known to globally interact with both messenger RNAs (mRNAs) and small regulatory RNAs (sRNAs). Here, we investigate ProQ-mRNA interactions in live bacteria using single-molecule tracking and reveal their dependence on the structural elements of ProQ. We illustrate that during exponential growth phase, stationary phase, and osmotic stress, ProQ mainly binds to mRNAs. All structural domains of ProQ, including N-terminal domain, linker region, and C-terminal domain, contribute to mRNA binding. While residue Y70 is generally required for global mRNA binding, other tested residues in the N-terminal domain have minor to moderate effects on mRNA binding, indicating that different mRNAs likely depend on different ProQ residues for binding. Finally, our data reveal that the expressed sRNA substrates have limited impacts on the fraction of mRNA-associated ProQ, likely due to the high abundance of mRNA-free ProQ inside the cell.