Genetic evidence that transcription activation by RhaS involves specific amino acid contacts with sigma 70
Genetic evidence that transcription activation by RhaS involves specific amino acid contacts with sigma 70
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DOI：
10.1128/jb.182.17.4959-4969.2000
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发表时间：
2000-09-01
影响因子：
3.2
通讯作者：
Egan, SM
中科院分区：
文献类型：
--
作者：
Bhende, PM;Egan, SM
RhaS响应于L-鼠李糖激活大肠杆菌rhaBAD和rhaT操纵子的转录，并且是转录激活因子的AraC/XylS家族的成员。我们希望确定sigma（70）是否可能是RhaS的活化靶。我们发现sigma（70）K593和R599似乎对rhaBAD和rhaT处的RhaS活化都很重要，但仅在缺乏第二种活化剂CRP的结合位点的截短启动子处。为了确定这些带正电荷的σ（70）残基是否可能接触RhaS，我们在RhaS的C-末端结构域中的带负电荷的残基处构建丙氨酸取代。在四个RhaS残基E181 A、D182 A、D186 A和D241 A处的置换在两个截短的启动子处都是缺陷的。最后，我们分析了RhaS和sigma（70）取代的组合，发现RhaS D241和sigma（70）R599满足在两个启动子处相互作用残基的标准。分子模拟表明，sigma（70）R599位于非常接近RhaS D241的位置;因此，这项工作提供了AraC/XylS家族蛋白中可能接触sigma（70）的特定残基的第一个证据。超过50%的AraC/XylS家族成员在RhaS D241的位置处具有Asp或Glu，表明这种与σ（70）的相互作用可能是保守的。
RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether sigma(70) might be an activation target for RhaS, We found that sigma(70) K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged sigma(70) residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and sigma(70) substitutions and found that RhaS D241 and sigma(70) R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that sigma(70) R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact sigma(70). More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with sigma(70) may be conserved.