Shi Y B, Gamper H, Hearst J E
Department of Chemistry, University of California, Berkeley 94720.
J Biol Chem. 1988 Jan 5;263(1):527-34.
We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques. The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end. The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct. Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion. The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints. In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex. These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex.
我们使用DNase I足迹技术，探究了T7 RNA聚合酶与DNA在延伸复合物中的相互作用，该延伸复合物被特意放置的补骨脂素双加合物或呋喃糖苷单加合物阻滞。补骨脂素衍生物HMT（4'-羟基甲基-4,5',8-三甲基补骨脂素）首先被放置在化学合成的双链DNA片段中间的一个特定位点，该片段一端含有T7 RNA聚合酶启动子。补骨脂素分子通过光化学作用，以双加合物形式连接到相反链上相邻的两个胸腺嘧啶残基，或以呋喃侧单加合物形式连接到编码链上仅一个胸腺嘧啶残基。以这些补骨脂素修饰的DNA作为转录模板，我们发现T7 RNA聚合酶在补骨脂素加合物位点被阻滞，并且停滞的延伸复合物保护编码链上位于加合物上游约15个核苷酸以及非编码链上围绕加合物的约20个核苷酸不被DNase I消化。两种补骨脂素修饰的DNA模板产生相同的RNA转录本和DNase I足迹。相比之下，T7聚合酶在起始复合物中仅保护从-20到+8的编码链。这些结果表明，RNA聚合酶从起始复合物转变为延伸复合物时会发生显著的构象变化。
