Ross W, Ernst A, Gourse R L
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Genes Dev. 2001 Mar 1;15(5):491-506. doi: 10.1101/gad.870001.
The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding. On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.
大肠杆菌RNA聚合酶（RNAP）的α亚基通过与富含A+T的上游元件（UP元件）结合，在识别许多启动子中发挥重要作用。UP元件是位于σ亚基识别元件（-35和-10六聚体）上游的一段DNA序列。我们使用高分辨率干涉和保护足迹法以及使用小沟结合药物Distamycin来研究DNA-RNAP相互作用。我们的结果表明，α亚基与DNA小沟中的碱基以及沿着小沟的DNA主链相互作用，但UP元件的大沟表面对α亚基的结合没有显著贡献。基于这些以及之前的结果，我们提出了一个模型，其中α亚基通过位于一对螺旋-发夹-螺旋基序中的氨基酸残基与UP元件DNA接触。此外，我们的实验通过鉴定-35和-10六聚体大沟中的功能基团（其中修饰会干扰RNAP结合），扩展了关于σ70对核心启动子识别的现有信息。这些研究极大地提高了我们对启动子-RNAP相互作用图景的分辨率。
