A non‐native C‐terminal extension of the β’ subunit compromises RNA polymerase and Rho functions

Escherichia coli RfaH abrogates Rho‐mediated polarity in lipopolysaccharide core biosynthesis operons, and ΔrfaH cells are hypersensitive to antibiotics, bile salts, and detergents. Selection for rfaH suppressors that restore growth on SDS identified a temperature‐sensitive mutant in which 46 C‐terminal residues of the RNA polymerase (RNAP) β’ subunit are replaced with 23 residues carrying a net positive charge. Based on similarity to rpoC397, which confers a temperature‐sensitive phenotype and resistance to bacteriophages, we named this mutant rpoC397*. We show that SDS resistance depends on a single nonpolar residue within the C397* tail, whereas basic residues are dispensable. In line with its mimicry of RfaH, C397* RNAP is resistant to Rho but responds to pause signals, NusA, and NusG in vitro similarly to the wild‐type enzyme and binds to Rho and Nus factors in vivo. Strikingly, the deletion of rpoZ, which encodes the ω “chaperone” subunit, restores rpoC397* growth at 42°C but has ..., Method is available in the published article., , # A non‐native C‐terminal extension of the β’ subunit compromises RNA polymerase and Rho functions

[https://doi.org/10.5061/dryad.ghx3ffbvg](https://doi.org/10.5061/dryad.ghx3ffbvg)

Mass-Spec of RpoC397\* cross-linking datasets.

There are two files in the dataset. Both files can be loaded into Scaffold (https://www.proteomesoftware.com/products/scaffold-5) for visualization and editing. The two files represent the two independent repeats.

大肠杆菌（Escherichia coli）RfaH蛋白可阻断Rho介导的脂多糖核心生物合成操纵子转录极性，且rfaH基因缺失（ΔrfaH）菌株对抗生素、胆汁盐与去污剂呈超敏表型。通过筛选可恢复菌株在十二烷基硫酸钠（SDS）培养基上生长的rfaH抑制突变株，我们获得一株温度敏感突变体：该突变体中RNA聚合酶（RNA polymerase, RNAP）β'亚基的46个C端残基被23个携带净正电荷的残基所取代。鉴于该突变体与赋予温度敏感表型及噬菌体抗性的rpoC397同源，我们将其命名为rpoC397*。我们的研究表明，SDS抗性取决于C397*尾部的单个非极性残基，而碱性残基对此并非必需。与RfaH的模拟效应一致，C397* RNAP对Rho具有抗性，但在体外对暂停信号、NusA及NusG的响应与野生型酶相似，且在体内可结合Rho与Nus因子。值得注意的是，编码ω“分子伴侣”亚基的rpoZ基因缺失可恢复rpoC397*菌株在42℃下的生长，但原文未详述后续内容，详细实验方法见已发表论文。
# β'亚基的非天然C端延伸损害RNA聚合酶与Rho的功能
[https://doi.org/10.5061/dryad.ghx3ffbvg]
RpoC397*交联实验质谱数据集
本数据集包含两个文件，均可导入Scaffold（https://www.proteomesoftware.com/products/scaffold-5）进行可视化与编辑，二者分别对应两次独立重复实验。