School of Microbiology and Immunology, University of Melbournegrid.1008.9, Melbourne, Victoria, Australia.
Mercian Corporation, Tokyo, Japan.
J Bacteriol. 2021 Sep 8;203(19):e0025221. doi: 10.1128/JB.00252-21.
A novel selection was developed for mutants of the C-terminal domain of RpoA (α-CTD) altered in activation by the TyrR regulatory protein of Escherichia coli K-12. This allowed the identification of an aspartate to asparagine substitution at residue 250 (DN250) as an activation-defective (Act) mutation. Amino acid residues known to be close to D250 were altered by mutagenesis, and the substitutions DR250, RE310, and RD310 were all shown to be defective in activation. None of these mutations caused defects in regulation of the upstream promoter (UP) element. The mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. The TyrR mutations EK139 and RG119 caused partial suppression of DN250, and TyrR RC119, RL119, RP119, RA77, and SG100 caused partial suppression of RE310. Additional activation-defective mutants (DT250, RS310, and EG288) were also isolated, using the chromosomal DN250 strain. Several new Act mutants were isolated in an strain, adding positions R77, D97, K101, D118, R119, R121, and E141 to known residues S95 and D103 and defining the activation patch on the amino-terminal domain (NTD) of TyrR. These results support a model for activation of TyrR-regulated genes where the activation patch on the TyrR NTD interacts with the TyrR-specific patch on the α-CTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants as activator contact sites. Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore, TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence and important plant interactions.
开发了一种新的选择方法，用于鉴定大肠杆菌 K-12 的 TyrR 调节蛋白改变了 C 端结构域（α-CTD）的 RpoA 突变体。这使得鉴定出天冬氨酸残基 250（D250）突变为天冬酰胺（DN250）为激活缺陷（Act）突变成为可能。通过诱变改变了已知接近 D250 的氨基酸残基，DR250、RE310 和 RD310 的突变都显示出激活缺陷。这些突变均未导致启动子上游元件（UP 元件）调节缺陷。将突变 DN250 转移到染色体上，以方便分离抑制突变。TyrR 突变 EK139 和 RG119 部分抑制了 DN250，而 TyrR RC119、RL119、RP119、RA77 和 SG100 部分抑制了 RE310。使用染色体 DN250 菌株，还分离出了其他一些新的激活缺陷突变体（DT250、RS310 和 EG288）。在一个突变体菌株中分离出了几种新的 Act 突变体，增加了 R77、D97、K101、D118、R119、R121 和 E141 等位置，这些位置在已知的 S95 和 D103 残基上定义了 TyrR 的氨基末端结构域（NTD）上的激活补丁。这些结果支持 TyrR 调节基因激活的模型，其中 TyrR NTD 上的激活补丁与 RNA 聚合酶的 TyrR 特异性补丁相互作用。鉴于已知的结构，这两个位点似乎都暴露在表面，提示 TyrR 激活的模型。它们还有助于解决文献中令人困惑的结果，这些结果表明 261 和 265 决定簇内的残基是激活剂接触位点。RNA 聚合酶对转录的调节是适应不断变化的环境和细胞分化的基础，这在所有生命领域都是如此。大肠杆菌中的基因是一个特别有用的模型，因为它通过一系列机制参与了大量操纵子的激活和抑制，并且与所有三种芳香族氨基酸以及可能的其他效应物相互作用。此外，TyrR 在许多其他属中也有同源物，调节许多不同的基因，利用不同的效应分子，在某些情况下影响毒力和重要的植物相互作用。
