A unique binding between SspA and RNAP β(')NTH across low-GC Gram-negative bacteria facilitates SspA-mediated transcription regulation.
A unique binding between SspA and RNAP β(')NTH across low-GC Gram-negative bacteria facilitates SspA-mediated transcription regulation.
复制标题
DOI：
10.1016/j.bbrc.2021.10.048
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Lin Wei
中科院分区：
文献类型：
--
作者：
Wang Fulin;Feng Yu;Shang Zhuo;Lin Wei
严格饥饿蛋白A（stringent starvation protein A，SspA）是一种转录因子，通过与大肠杆菌RNA聚合酶（EcoRNAP）β′亚基的σ 70区域4和锌结合域（zinc binding domain，ZBD）相互作用，调控σ70依赖性基因的转录。尽管最近报道了广泛的生化和结构分析，但SspA与RNAP的相互作用尚未得到全面了解。在这里，我们重新处理了我们之前的EcoRNAP启动子与SspA开放复合物（SspA-RPo）的冷冻EM数据集，并获得了显着改善的密度图。出乎意料的是，新图谱显示SspA与β′亚基（β′ NTH）和ω亚基的N-末端螺旋相互作用，这有助于稳定SspA-EcoRNAP σ 70全酶复合物。对不同种类细菌β′亚基N端序列的比对和系统发育树分析表明，β′ NTH高度保守，仅存在于携带SspA的低GC含量革兰氏阴性菌中，表明β′ NTH与SspA存在协同进化关系。野生型SspA及其突变体的转录测定表明，SspA与β′ NTH之间的相互作用促进了SspA的转录调控。总之，我们的研究结果提供了一个更全面的了解SspA和RNAP之间的相互作用，以及它们在细菌转录调控中的作用。
Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate σ70-dependent gene transcription through interacting with σ70region 4 and the zinc binding domain (ZBD) ofE. coliRNA polymerase (EcoRNAP) β′ subunit simultaneously. Despite extensive biochemical and structural analyses were reported recently, the interactions of SspA with RNAP are not comprehensively understood. Here, we reprocessed our previous cryo-EM dataset ofEcoRNAP-promoter open complex with SspA (SspA-RPo) and obtained a significantly improved density map. Unexpectedly, the new map showed that SspA interacts with both N-terminal helix of β′ subunit (β′ΝΤΗ) and ω subunit, which contributes to stabilize the SspA-EcoRNAP σ70holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of β′ subunit from different classes of bacteria revealed that β′ΝΤΗ is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of β′ΝΤΗ and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and β′ΝΤΗ facilitates the transcription regulation of SspA. Together, our results provide a more comprehensive insight into the interactions between SspA and RNAP and their roles in bacterial transcription regulation.