Institute of Molecular Infection Biology (IMIB), University of Würzburg, D-97080 Würzburg, Germany.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), D-97080 Würzburg, Germany.
RNA. 2021 Dec;27(12):1512-1527. doi: 10.1261/rna.078954.121. Epub 2021 Sep 8.
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 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.
FinO 结构域蛋白 ProQ 属于一种广泛存在的 RNA 结合蛋白（RBPs）家族，参与细菌染色体和移动元件中的基因调控。虽然 ProQ 在不同细菌中的细胞 RNA 靶标已经确定，但在生理条件下，功能关键的 ProQ 残基仍有待鉴定。在发现 ProQ 缺乏可缓解 仅以琥珀酸作为唯一碳源时的生长抑制后，我们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出多个 ProQ 残基，这些残基对于 ProQ 活性是必需的。两个羧基末端突变消除了 ProQ 的功能，并轻微损害了模型 RNA 靶标的结合。相比之下，FinO 结构域中的几个突变使 ProQ 既无功能又无法在体内与靶 RNA 相互作用。FinO 结构域的改变刺激了 ProQ 被 Lon 介导的蛋白水解迅速周转，这表明存在一种质量控制机制，可以防止无功能 ProQ 分子的积累。我们将这一观察结果扩展到了 Hfq，它是肠道细菌的另一种主要 sRNA 伴侣。在 RNA 结合和寡聚化方面有缺陷的 Hfq Y55A 突变蛋白不稳定，容易被 Lon 降解。总之，我们的发现将主要的 AAA+ 家族蛋白酶 Lon 与 Hfq 和 ProQ 的 RNA 依赖性质量控制联系起来，Hfq 和 ProQ 是革兰氏阴性菌的两种主要 sRNA 伴侣。
