Bogacz Marek, El Abbar Faiha M, Cox Claudia A, Li Jiaqi, Fiedler Jarred S, Tran Lynn K H, Tran Paul M H, Daugherty C Luke, Blake Kate H, Wang Zhirui, Azadi Parastoo, Thompson Stuart A
Division of Infectious Diseases, Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA, United States.
Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, United States.
Front Microbiol. 2021 Jan 11;11:531596. doi: 10.3389/fmicb.2020.531596. eCollection 2020.
CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.
CsrA是一种mRNA结合的转录后调节因子，它控制着这种重要病原体的许多与代谢和毒力相关的特性。与CsrA不同，后者的活性通过与小非编码RNA（sRNA）结合来调节，而CsrA的活性则通过与CsrA拮抗剂FliW结合来控制。在本研究中，我们确定了CsrA上的FliW结合位点。删除CsrA的C末端（相对于与sRNA结合的CsrA蛋白有所延长）消除了FliW的结合。细菌双杂交实验用于评估FliW与野生型CsrA及其突变体的相互作用，其中每个氨基酸都被单独突变。两个CsrA突变（V51A和N55A）导致FliW结合显著减少。通过尺寸排阻色谱法和表面等离子体共振评估，V51A和N55A突变体还显示CsrA-FliW复合物形成减少。这些残基在含有预测其活性受FliW调节的CsrA直系同源物的细菌物种中高度保守。FliW结合的位置紧邻CsrA同二聚体的两个RNA结合位点，这表明FliW与CsrA结合通过空间位阻、静电排斥或改变RNA结合位点的整体结构来调节其与mRNA靶标的结合能力的模型。
