Shi Y B, Gamper H, Van Houten B, Hearst J E
Department of Chemistry, University of California, Berkeley 94720.
J Mol Biol. 1988 Jan 20;199(2):277-93. doi: 10.1016/0022-2836(88)90314-2.
We have probed the interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested by a site-specifically placed psoralen crosslink using DNase I footprinting techniques. The psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing an E. coli RNA polymerase promoter at one end. The psoralen molecule was photochemically attached to two adjacent thymidine residues on opposite strands as a diadduct. Using this crosslinked DNA as the template for transcription, we found that the E. coli RNA polymerase was blocked at the psoralen diadduct, yielding a transcript 29 nucleotides long. The arrested elongation complex inhibited DNase I digestion of both the coding strand and the non-coding strand from about 22 nucleotides upstream to 15 nucleotides downstream from the diadduct. These results, which suggest that the unwindase and the catalytic sites of the polymerase are very close to each other, have been incorporated into a model of the transcription elongation complex.
我们使用DNase I足迹技术，研究了在由位点特异性放置的补骨脂素交联所阻滞的延伸复合物中，大肠杆菌RNA聚合酶与DNA的相互作用。补骨脂素衍生物4'-羟甲基-4,5',8-三甲基补骨脂素首先被放置在一个化学合成的双链DNA片段中间的特定位点，该片段一端含有一个大肠杆菌RNA聚合酶启动子。补骨脂素分子通过光化学作用作为双加合物连接到相反链上的两个相邻胸腺嘧啶残基上。以这种交联的DNA作为转录模板，我们发现大肠杆菌RNA聚合酶在补骨脂素双加合物处受阻，产生了一个29个核苷酸长的转录本。停滞的延伸复合物抑制了从双加合物上游约22个核苷酸到下游15个核苷酸处编码链和非编码链的DNase I消化。这些结果表明解旋酶和聚合酶的催化位点彼此非常接近，并已被纳入转录延伸复合物的模型中。
