转录复合物与补骨脂素的光化学交联。I. 体外SV40新生RNA与其双链DNA模板的共价连接。
Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV40 nascent RNA to its double-stranded DNA template.
作者信息
Shen C K, Hearst J E
出版信息
Nucleic Acids Res. 1978 Apr;5(4):1429-41. doi: 10.1093/nar/5.4.1429.
Abstract
14C-labeled SV40 DNA has been transcribed with E. coli RNA polymerase using 3H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT), at 37 degrees C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) SV40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not required for successful crosslinkage.
摘要
利用3H标记的核糖核苷三磷酸作为前体，用大肠杆菌RNA聚合酶对14C标记的SV40 DNA进行了转录。然后将所得的转录复合物在37℃下用光化学方法与补骨脂素衍生物4'-氨甲基-4,5',8-三甲基补骨脂素（AMT）进行交联，并在SDS-蔗糖梯度中进行分析。发现在变性蔗糖梯度中，光化学交联过程使新生RNA链与其双链（螺旋状）SV40模板共同沉降。这一结果以及几个对照实验表明，光化学反应后新生RNA与螺旋状DNA之间形成了共价连接。观察到交联现象与用作模板的DNA的超螺旋状态无关。成功进行交联不需要事先添加EDTA来终止转录。
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