Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus
Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus
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DOI：
10.1128/jb.175.9.2652-2661.1993
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发表时间：
1993-05
影响因子：
3.2
通讯作者：
D. Vujaklija;S. Horinouchi;T. Beppu
中科院分区：
文献类型：
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作者：
D. Vujaklija;S. Horinouchi;T. Beppu
利用迁移率转换聚丙烯酰胺凝胶电泳的DNA结合试验揭示了一种蛋白质的存在，该蛋白质特异性地结合到strR转录起始点上游-288到-191 bp的限制性片段，strR是灰色链霉菌中链霉素生物合成的调控基因。结合位点对应于从体内启动子测定的结果预测的上游激活序列。5 mM Mg 2+大大增强了结合。仅用来自野生型菌株的蛋白质来源检测到这种结合，而不是来自A因子缺陷突变菌株。外源性补充的A-因子的A-因子缺陷的突变株引起的DNA结合试验中的蛋白质的外观。一个长度为52 bp的合成核苷酸（区域从-293到-242），它是根据从切割DNA片段的阻滞试验和体内启动子试验获得的数据合成的，被A因子依赖性蛋白阻滞。除了这种A-因子依赖性蛋白质，至少有三种蛋白质具有不同的识别位点的亲和力，能够结合到上游区域的strR启动子进行检测。在孵育混合物中存在ATP和Mg 2+时，这些蛋白质之一与A因子依赖性蛋白结合的上游激活序列的两侧的结合被完全废除。与这些蛋白质结合的区域显示出异常的电泳迁移率，就像弯曲的DNA分子一样，这可能是由A和T组成的许多块的存在引起的。这些蛋白质结合的区域被发现在相反的方向上的strR转录。
DNA-binding assays using mobility shift polyacrylamide gel electrophoresis revealed the presence of a protein that specifically bound to a restriction fragment -288 to -191 bp upstream from the transcriptional start point of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus. The binding site corresponded to an upstream activation sequence predicted from the results of in vivo promoter assays. The binding was greatly enhanced by 5 mM Mg2+. This binding was detected with the protein source only from the wild-type strain and not from an A-factor-deficient mutant strain. The exogenous supplementation of A-factor to the A-factor-deficient mutant strain caused the appearance of the protein in the DNA-binding assay. A synthetic nucleotide 52 bp in length (region from -293 to -242), which was synthesized on the basis of data obtained from both retardation assays with dissected DNA fragments and in vivo promoter assays, was retarded by the A-factor-dependent protein. In addition to this A-factor-dependent protein, at least three proteins with different recognition site affinities capable of binding to the upstream region of the strR promoter were detected. The binding of one of these proteins to both sides of the upstream activation sequence bound by the A-factor-dependent protein was completely abolished in the presence of ATP and Mg2+ in the incubation mixture. The region bound by these proteins showed anomalous electrophoretic mobility, like that of a bent DNA molecule, which is probably caused by the presence of many blocks consisting of A and T. The region bound by these proteins was found to be transcribed in the orientation opposite to that of strR.