Construction and functional analyses of a comprehensive sigma54 site-directed mutant library using alanine-cysteine mutagenesis.
Construction and functional analyses of a comprehensive sigma54 site-directed mutant library using alanine-cysteine mutagenesis.
复制标题
使用丙氨酸-半胱氨酸诱变构建综合 sigma54 定点突变体库并进行功能分析。
DOI：
10.1093/nar/gkp419
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发表时间：
2009
影响因子：
14.9
通讯作者：
Xiao Y
中科院分区：
文献类型：
--
作者：
Xiao Y
σ54 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，否则无法启动转录。 σ54 密切参与激活剂依赖性转录的许多步骤，例如核心 RNAP 结合、启动子识别、激活剂相互作用和开放复合物形成。为了系统地定义有助于每个功能的 σ54 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完整的 σ54 突变体库。 Cys(-)σ54的3至476位氨基酸残基一次以两个相邻残基为一组系统地突变为丙氨酸和半胱氨酸。体内外分析了各取代对对σ54功能的影响，并首次揭示了许多残基的功能。 σ54异构化活性的增加很少与全酶转录活性的增加相对应，这表明σ54异构化后的步骤（可能是核心RNAP结构的变化）也受到严格调节或速率限制以形成开放复合物。核心 RNAP 结合活性和激活剂响应性之间的联系表明 σ54-核心 RNAP 界面在激活时发生变化。
The σ54factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. σ54is 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 σ54residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of σ54was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)σ54were 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 σ54were analyzedin vivoandin vitroand the functions of many residues were revealed for the first time. Increased σ54isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after σ54isomerization, 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 σ54-core RNAP interface changes upon activation.