Program in Biochemistry, Mount Holyoke College, South Hadley, MA 01075, USA.
Department of Biological Sciences, Mount Holyoke College, South Hadley, MA 01075, USA.
Nucleic Acids Res. 2020 May 7;48(8):4507-4520. doi: 10.1093/nar/gkaa144.
The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.
FinO 结构域蛋白 ProQ 是一种 RNA 结合蛋白，已知在变形菌中发挥渗透调节作用。最近，ProQ 已被证明在沙门氏菌和大肠杆菌中作为一种全局 RNA 结合蛋白发挥作用，与数十种小 RNA（sRNA）和信使 RNA（mRNA）结合，通过与 5'和 3'非翻译区（UTR）的相互作用来调节 mRNA 表达水平。尽管 ProQ 作为一种新型的全局 RNA 结合蛋白及其作为配体 RNA 伴侣的潜力令人兴奋，但我们对 ProQ 与 RNA 相互作用的分子机制仍存在很大的理解空白。为了将分子遗传学工具应用于这个问题，我们已经对细菌三杂交（B3H）测定法进行了改编，以检测 ProQ 与靶 RNA 的相互作用。通过结构域截断、定点突变和无偏正向遗传筛选，我们确定了 ProQ 的 NTD 上一组高度保守的残基是体内识别两种 RNA 的主要表面，并提出 NTD 结构作为识别 RNA 双链形状的静电支架。
