Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease
The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria. SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.