Lesley S A, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Biochemistry. 1989 Sep 19;28(19):7728-34. doi: 10.1021/bi00445a031.
A set of internal deletions and frame-shift mutations was made in the structural gene for the major sigma factor of Escherichia coli RNA polymerase (sigma 70). The truncated proteins from these various mutants were examined to determine if they retained the ability to bind core RNA polymerase. Two assays were used to determine core-binding activity. Gel filtration was used to separate free sigma 70 from sigma 70 bound to core polymerase. Immunoprecipitation of polymerase using an anti-alpha-subunit monoclonal antibody was also used to determine if the various truncated proteins were bound to core. Results from these experiments indicate core-binding activity is retained when large portions of the sigma 70 protein are deleted. Deletion of a region in the central portion of the protein caused a large decrease in core-binding activity. The results suggest that the region spanning amino acids 361-390 is important for efficient core-binding activity. Sequence comparison of various sigma factors shows highly conserved amino acids in this region. A synthetic peptide having the sequence of amino acids 361-390 was synthesized and examined for the ability to bind core RNA polymerase.
在大肠杆菌RNA聚合酶主要σ因子（σ70）的结构基因中制造了一组内部缺失和移码突变。对这些不同突变体产生的截短蛋白进行检测，以确定它们是否保留与核心RNA聚合酶结合的能力。使用了两种测定方法来确定核心结合活性。凝胶过滤用于将游离的σ70与结合到核心聚合酶上的σ70分离。使用抗α亚基单克隆抗体对聚合酶进行免疫沉淀，也用于确定各种截短蛋白是否与核心结合。这些实验结果表明，当σ70蛋白的大部分被缺失时，仍保留核心结合活性。蛋白质中部区域的缺失导致核心结合活性大幅下降。结果表明，跨越氨基酸361 - 390的区域对于有效的核心结合活性很重要。各种σ因子的序列比较显示该区域存在高度保守的氨基酸。合成了具有氨基酸361 - 390序列的合成肽，并检测其结合核心RNA聚合酶的能力。
