The RNA-binding protein CsrA is a post-transcriptional regulator that is encoded in genomes throughout the bacterial phylogeny. In the gamma-proteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, that non-competitively inhibits CsrA activity but the precise mechanism of antagonism is unclear. Here we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition that fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues that surround the critical regulatory CsrA residue N55 and abolished CsrA interaction. Two loss-of-function alleles however mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of these two alleles reversed charge at what appeared to be a salt bridge with the CsrA core region, charge reversal of the CsrA partner residue phenocopied the FliW allele, and charge reversal of both residues simultaneously restored antagonism. We propose a model in which initial interaction between FliW and CsrA is necessary but not sufficient for antagonism which also requires salt bridge formation with, and deformation of, the CsrA core domain to allosterically abolish RNA binding activity.

中文翻译：

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FliW对枯草芽孢杆菌的变构抑制作用需要与CsrA核心接触

RNA结合蛋白CsrA是一种转录后调节剂，在整个细菌系统发育过程中的基因组中都有编码。在γ-变形细菌中，CsrA的活性受到竞争性螯合CsrA结合的小RNA的抑制。相反，坚硬的枯草芽孢杆菌编码一种称为FliW的CsrA蛋白抑制剂，该抑制剂非竞争性地抑制CsrA活性，但拮抗作用的确切机制尚不清楚。在这里，我们采用无偏遗传方法来鉴定对CsrA抑制重要的FliW残基，该残基分为两个不同的空间和功能类别。大多数功能丧失的等位基因突变了关键调控CsrA残基N55周围的FliW残基，并废除了CsrA相互作用。但是，两个功能丧失的等位基因突变了CsrA核心二聚化结构域附近的FliW残基，并维持了与CsrA的相互作用。这两个等位基因之一在与CsrA核心区域形成盐桥的情况下使电荷反向，CsrA伴侣残基的电荷反向表型化了FliW等位基因，而两个残基的电荷反向同时恢复了拮抗作用。我们提出了一个模型，其中FliW和CsrA之间的初始相互作用是必要的，但对于拮抗作用还不够，拮抗作用还要求与CsrA核心结构域形成盐桥并使其变形以变构地消除RNA结合活性。和两个残基的电荷逆转同时恢复了拮抗作用。我们提出了一个模型，其中FliW和CsrA之间的初始相互作用是必要的，但不足以拮抗作用，而拮抗作用还需要与CsrA核心结构域形成盐桥并使其变形以变构地消除RNA结合活性。和两个残基的电荷逆转同时恢复了拮抗作用。我们提出了一个模型，其中FliW和CsrA之间的初始相互作用是必要的，但不足以拮抗作用，而拮抗作用还需要与CsrA核心结构域形成盐桥并使其变形以变构地消除RNA结合活性。