Zandomeni R, Bunick D, Ackerman S, Mittleman B, Weinmann R
J Mol Biol. 1983 Jul 5;167(3):561-74. doi: 10.1016/s0022-2836(83)80098-9.
5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analogue, has been used previously as an inhibitor of heterogeneous nuclear and messenger RNA synthesis. In an in vitro transcriptional system, we have detected inhibition of synthesis of full-length runoff RNAs at concentrations at which in vivo mRNA synthesis is inhibited. By hybridization of RNA synthesized in vitro to single-stranded DNA and gel analysis, we were able to reduce the background of the transcription reaction, detect DRB-induced inhibition of full-length runoff RNAs and DRB-insensitive transcription of short RNAs. To establish further the effect of DRB on initiation of transcription, preincubation experiments with template, whole cell extract and two initial nucleotides of the transcript were performed. Elongation was then measured as discrete-sized RNAs transcribed from the truncated template after addition of the other triphosphates (one of them labeled), in the presence or absence of DRB. An effect on initiation but not on elongation or termination was detected. Fingerprint analysis of these runoff RNAs indicates that the labeling of U in the presence of DRB is uniform throughout the molecule. A model to explain a novel interpretation of the action of DRB is presented.
5,6-二氯-1-β-D-呋喃核糖基苯并咪唑是一种腺苷类似物，此前已被用作异质核RNA和信使RNA合成的抑制剂。在体外转录系统中，我们发现在体内mRNA合成受到抑制的浓度下，全长延伸RNA的合成也受到了抑制。通过将体外合成的RNA与单链DNA杂交并进行凝胶分析，我们能够降低转录反应的背景，检测到DRB对全长延伸RNA的抑制作用以及DRB不敏感的短RNA转录。为了进一步确定DRB对转录起始的影响，我们进行了模板、全细胞提取物和转录本的两个起始核苷酸的预孵育实验。然后在添加其他三磷酸核苷酸（其中一种被标记）后，在有或没有DRB的情况下，将延伸测量为从截短模板转录的离散大小的RNA。检测到对起始有影响，但对延伸或终止没有影响。对这些延伸RNA的指纹分析表明，在DRB存在下U的标记在整个分子中是均匀的。本文提出了一个模型来解释对DRB作用的一种新解释。
