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. While 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 amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In 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 nonfunctional 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.

中文翻译：

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RNA 结合蛋白 ProQ 的扫描诱变揭示了 Lon 蛋白酶的质量控制作用


FinO 结构域蛋白 ProQ 属于广泛分布的 RNA 结合蛋白 (RBP) 家族，参与细菌染色体和移动元件的基因调控。虽然 ProQ 的细胞 RNA 靶标已在多种细菌中建立，但在生理条件下功能至关重要的 ProQ 残基仍有待鉴定。我们发现 ProQ 缺陷可以减轻以琥珀酸作为唯一碳源的沙门氏菌的生长抑制，因此设计了一种实验进化方法来利用这种表型。通过将突变扫描与功能丧失选择相结合，我们在氨基末端 FinO 结构域和可变羧基末端区域中鉴定了 ProQ 活性所需的多个 ProQ 残基。两个羧基末端突变消除了 ProQ 功能，并轻度损害了模型 RNA 靶标的结合。相比之下，FinO 结构域中的几个突变使 ProQ 功能失活并且无法与体内靶 RNA 相互作用。 FinO 结构域的改变通过 Lon 介导的蛋白水解作用刺激了 ProQ 的快速周转，这表明存在一种防止非功能性 ProQ 分子积累的质量控制机制。我们将这一观察扩展到 Hfq，这是肠道细菌的另一个主要 sRNA 伴侣。 Hfq Y55A 突变蛋白在 RNA 结合和寡聚化方面存在缺陷，被证明不稳定且容易被 Lon 降解。综上所述，我们的研究结果将主要的 AAA+ 家族蛋白酶 Lon 与 Hfq 和 ProQ（革兰氏阴性菌的两种主要 sRNA 伴侣）的 RNA 依赖性质量控制联系起来。