Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.
Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.
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RNA 聚合酶 sigma(70) 区域 4 和大肠杆菌 SoxS（一种通过预募集机制发挥作用的转录激活剂）之间的蛋白质-蛋白质相互作用：“DNA 外”和“DNA 上”相互作用的证据。
DOI：
10.1016/j.jmb.2010.05.052
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发表时间：
2010
影响因子：
5.6
通讯作者：
WolfJr,RichardE
中科院分区：
文献类型：
--
作者：
Zafar,MAmmar;Shah,IshitaM;WolfJr,RichardE
根据招募前假设，大肠杆菌SoxS通过与RNA聚合酶（RNAP）形成二元复合物激活SoxRS调控基因的转录，RNAP扫描染色体寻找SoxRS依赖性I类和II类启动子。我们之前已经证明，α亚基的c端结构域通过与SoxS进行蛋白-蛋白接触来激活这两类启动子；其中一些接触是在没有启动子DNA的情况下在溶液中进行的，这是对招募前假设的关键预测。本研究发现，在RNAP的σ70区（σ70R4）中，有7个单丙氨酸取代降低了SoxS对II类启动子的激活。通过对这些σ70R4突变体与SoxS阳性对照突变体的遗传上位性测试，我们在大肠杆菌中发现了10对相互作用的氨基酸。通过酵母双杂交系统和亲和固定实验，我们发现SoxS和σ70R4可以在溶液中相互作用（即“off-DNA”）。这种相互作用需要SoxS的I/II类（而不是II类）阳性对照面的氨基酸，并且在大肠杆菌中降低活性的5个σ70R4氨基酸也降低了SoxS -σ70R4在酵母中的相互作用。发生在大肠杆菌中的上位性相互作用之一也发生在酵母双杂交系统中（即off-DNA）。重要的是，我们推断大肠杆菌中需要II类表面氨基酸的五种上位相互作用发生在II类启动子的“dna上”。发现SoxS在dna外和dna上均与σ70R4接触，这与招募前假设一致。此外，SoxS现在是大肠杆菌转录激活子的第一个例子，它使用单个阳性对照表面与RNAP的两个不同亚基进行特定的蛋白质-蛋白质接触。
According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the α subunit's C-terminal domain plays a role in activating both classes of promoter by making protein–protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of σ70(σ70R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these σ70R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and σ70R4 can interact in solution (i.e., “off-DNA”). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of σ70R4 that reduce activation in E. coli also reduce the SoxS–σ70R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur “on-DNA” at class II promoters. Finding that SoxS contacts σ70R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein–protein contacts with two different subunits of RNAP.