Siegele D A, Hu J C, Walter W A, Gross C A
Department of Bacteriology, University of Wisconsin, Madison 53706.
J Mol Biol. 1989 Apr 20;206(4):591-603. doi: 10.1016/0022-2836(89)90568-8.
We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.
我们利用与37个突变型和野生型启动子的转录融合，系统地分析了编码大肠杆菌RNA聚合酶全酶σ70亚基的基因rpoD中13个突变的体内启动子识别特性。我们发现了三类rpoD突变：（1）表明σ70氨基酸侧链与启动子中特定碱基之间存在接触的突变；（2）似乎影响与启动子DNA的序列非依赖性接触或聚合酶异构化的突变；（3）对启动子识别几乎没有影响或没有影响的突变。我们的结果使我们提出，σ70 C末端附近的一个序列，类似于噬菌体和细菌DNA结合蛋白的螺旋-转角-螺旋DNA结合基序，负责识别-35区域，并且σ70内部的一个序列，在σ因子中高度保守的区域，识别启动子的-10区域。位于所提出的螺旋-转角-螺旋基序识别螺旋中的rpoD突变影响与-35区域中特定碱基的相互作用，而上游螺旋中的突变，被认为与磷酸主链接触，对启动子识别具有序列非依赖性影响。
