Mutational analysis of σ70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA
Mutational analysis of σ70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA
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DOI：
10.1016/j.jmb.2006.08.074
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发表时间：
2006-11-10
影响因子：
5.6
通讯作者：
Hinton, Deborah M.
中科院分区：
文献类型：
--
作者：
Baxter, Kimberly;Lee, Jennifer;Hinton, Deborah M.
噬菌体 T4 中间启动子的转录激活需要含有 sigma(70) 的大肠杆菌 RNA 聚合酶、T4 激活剂 MotA 和 T4 共激活剂 AsiA。 T4 中间启动子包含 sigma(70)-10 DNA 元件。然而，这些启动子缺乏 sigma(70)-35 元件，而是具有以 -30 为中心的 MotA 框，该框与 MotA 结合。先前的工作表明，AsLk 和 MotA 与 sigma(70) 的区域 4 相互作用，该区域是通常接触 -35 DNA 的 C 端部分和核心中的 β-flap 结构。 AsiA 结合可阻止 sigma(70)/beta-flap 和 sigma(70)/-35 DNA 相互作用，从而抑制需要 -35 元件的启动子的转录。为了测试区域 4 内的残基对于 MotA 和 AsiA 功能的重要性，我们研究了 sigma(70) 区域 4 突变体如何与 AsiA、MotA 和 beta-flap 相互作用以及在体外转录测定中的功能。我们发现残基584-588（区域4.2）处的丙氨酸取代不会损害区域4与β-flap或MotA的相互作用，但它们消除了与AsiA的相互作用并防止AsiA抑制和MotA/AsiA激活。相反，551-552、554-555（区域 4.1）处的丙氨酸取代消除了区域 4/β-flap 相互作用，显着损害了 AsiA/sigma(70) 相互作用，并消除了 AsiA 抑制。然而，如果 MotA 和 AsiA 都存在，4.1 突变体 sigma(70) 仍然完全能够激活。先前的 NMR 结构显示 AsiA 与 sigma(70) 区域 4 结合，显着扭曲区域 4.1 和 4.2，并间接改变 sigma(70) C 末端 MotA 相互作用位点的构象。我们的分析提供了结构中鉴定的 a 70 残基的生化相关性，表明 AsiA 与 sigma(70) 区域 4.2 的相互作用对于激活至关重要，并支持 AsiA 结合促进 MotA 与 sigma(70) 远 C 末端之间相互作用的观点。由 Elsevier Ltd 出版。
Transcriptional activation of bacteriophage T4 middle promoters requires sigma(70)-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the sigma(70)-10 DNA element. However, these promoters lack the sigma(70)-35 element, having instead a MotA box centered at -30, which is bound by MotA. Previous work has indicated that AsLk and MotA interact with region 4 of sigma(70), the C-terminal portion that normally contacts -35 DNA and the beta-flap structure in core. AsiA binding prevents the sigma(70)/beta-flap and sigma(70)/-35 DNA interactions, inhibiting transcription from promoters that require a -35 element. To test the importance of residues within a region 4 for MotA and AsiA function, we investigated how sigma(70) region 4 mutants interact with AsiA, MotA, and the beta-flap and function in transcription assays in vitro. We find that alanine substitutions at residues 584-588 (region 4.2) do not impair the interaction of region 4 with the beta-flap or MotA, but they eliminate the interaction with AsiA and prevent AsiA inhibition and MotA/AsiA activation. In contrast, alanine substitutions at 551-552, 554-555 (region 4.1) eliminate the region 4/beta-flap interaction, significantly impair the AsiA/sigma(70) interaction, and eliminate AsiA inhibition. However, the 4.1 mutant sigma(70) is Still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to sigma(70) region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the sigma(70) C terminus. Our analyses provide biochemical relevance for the a 70 residues identified in the structure, indicate that the interaction of AsiA with sigma(70) region 4.2 is crucial for activation, and support the idea that AsiA binding facilitates an interaction between MotA and the far C terminus of sigma(70) .Published by Elsevier Ltd.