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首页> 外文期刊>Nucleic Acids Research >Construction and functional analyses of a comprehensive set site-directed mutant library using alanine-cysteine mutagenesis.
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Construction and functional analyses of a comprehensive set site-directed mutant library using alanine-cysteine mutagenesis.

机译：使用丙氨酸-半胱氨酸诱变的综合设置的定点突变文库的构建和功能分析。

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摘要

The set factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. set is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define set residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of set was constructed by alanine-cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)set were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of set were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased set isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after set isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the set-core RNAP interface changes upon activation.
机译：设定因子与核心RNA聚合酶（RNAP）结合形成全酶，除非受到激活蛋白的作用，否则其无法启动转录。该集合紧密参与激活物依赖性转录的许多步骤，例如核心RNAP结合，启动子识别，激活物相互作用和开放复合物形成。为了系统地定义有助于这些功能中的每一个的集合残基并生成用于位点特异性蛋白质标记的资源，通过丙氨酸-半胱氨酸扫描诱变构建了一个完整的集合突变库。 Cys（-）set的3到476个氨基酸残基一次被系统突变为两个相邻残基组成的丙氨酸和半胱氨酸。在体内和体外分析了每个取代对对集合功能的影响，并首次揭示了许多残基的功能。集合异构化活性的提高很少与全酶的转录活性相对应，这表明集合异构化后的步骤（可能是核心RNAP结构的变化）也受到严格调节或限制了开放复合物的形成。核心RNAP结合活性和激活剂响应性之间的联系表明，设置核心的RNAP界面在激活时发生变化。

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