Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ
Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ
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DOI：
10.1073/pnas.0405883101
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发表时间：
2004-09-21
影响因子：
11.1
通讯作者：
Wahl, MC
中科院分区：
文献类型：
--
作者：
Bonin, I;Mühlberger, R;Wahl, MC
来自大肠杆菌的转录因子NusA的C端包含两个重复单元，它们在抗终止过程中与噬菌体λ的蛋白N结合。为了描述NusA-lambdaN相互作用的结构基础，我们试图结晶NusA c末端重复序列与lambdaN肽的复合物（残基34-47）。两个NusA结构域在结晶过程中被蛋白水解分离，晶体中含有与单个lambdaN片段接触的第一个重复单元的两个拷贝。NusA模块使用相同的区域接触肽，但从相反的侧面接近配体。与与boxB RNA复合物中lambdaN N端的α -螺旋构象相反，lambdaN的残基34-40在与NusA相互作用后仍然延长。突变分析表明，观察到的NusA-lambdaN相互作用模式中只有一种具有生物学意义，支持NusA和lambdaN在抗末端复合物中的等摩尔比例。溶液研究表明，第二个NusA重复单元促进了额外的相互作用，与已知的NusA和lambdaN的补偿性突变一致。与RNA聚合酶a亚基相反，lambdaN的结合不会刺激NusA的RNA相互作用。结果表明，在抗终止复合物中，lambdaN作为一个支架紧密地对抗NusA和mRNA。
The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase a subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.