Amino acid contacts between sigma 70 domain 4 and the transcription activators RhaS and RhaR.
Amino acid contacts between sigma 70 domain 4 and the transcription activators RhaS and RhaR.
复制标题
西格玛 70 结构域 4 和转录激活剂 RhaS 和 RhaR 之间的氨基酸接触。
DOI：
10.1128/jb.186.18.6277-6285.2004
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发表时间：
2004
影响因子：
3.2
通讯作者：
Egan,SusanM
中科院分区：
文献类型：
--
作者：
Wickstrum,JasonR;Egan,SusanM
RhaS和RhaR蛋白是转录激活剂，其响应l-鼠李糖的可用性并激活大肠杆菌-鼠李糖分解代谢调节子中操纵子的转录。 RhaR 激活rhaSR 的转录，RhaS 激活编码鼠李糖分解代谢酶rhaBAD 的操纵子以及编码鼠李糖转运蛋白rhaT 的操纵子的转录。 RhaS 与 RhaR 在氨基酸水平上有 30% 的相同性，并且两者都是转录激活子 AraC/XylS 家族的成员。 RhaS 和 RhaR 结合位点与它们所调节的启动子的 -35 六聚体重叠，表明它们可能接触 RNA 聚合酶的 σ70 亚基，作为其转录激活机制的一部分。为了支持这一假设，我们的实验室之前发现了 RhaS 残基 D241 和 σ70 残基 R599 之间的相互作用。在本研究中，我们首先鉴定了σ70、K593和R599中的两个带正电的氨基酸，以及RhaR、D276、E284和D285中的三个带负电的氨基酸，它们对于RhaR介导的therhaSRoperon转录激活很重要。使用遗传失去接触方法，我们获得了 RhaR D276 和 σ70R599 之间特定接触的证据。最后，我们实验室之前的结果分别表明，RhaS D250A 和 σ70K593A 在therhaBAD启动子上存在缺陷。我们对这些残基的遗传接触缺失分析表明，它们确定了 RhaS 和 σ70 之间的第二个接触位点。
The RhaS and RhaR proteins are transcription activators that respond to the availability ofl-rhamnose and activate transcription of the operons in theEscherichia colil-rhamnose catabolic regulon. RhaR activates transcription ofrhaSR, and RhaS activates transcription of the operon that encodes thel-rhamnose catabolic enzymes,rhaBAD, as well as the operon that encodes thel-rhamnose transport protein,rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the −35 hexamers of the promoters they regulate, suggesting they may contact the σ70subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and σ70residue R599. In the present study, we first identified two positively charged amino acids in σ70, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of therhaSRoperon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and σ70R599. Finally, previous results from our lab separately showed that RhaS D250A and σ70K593A were defective at therhaBADpromoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and σ70.