Campbell F E, Setzer D R
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4960.
Mol Cell Biol. 1992 May;12(5):2260-72. doi: 10.1128/mcb.12.5.2260-2272.1992.
Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.
非洲爪蟾RNA聚合酶III在没有转录起始通常所需的其他III类转录因子的情况下，能在poly(dC)尾DNA模板上特异性地起始转录。在使用5S rRNA基因位于尾端下游的DNA片段进行转录终止的实验分析中，只有40%的正在转录的聚合酶分子在正常高效的非洲爪蟾体细胞型5S rRNA终止子处终止；其余60%的分子会通读这些信号并产生延伸转录本。我们发现，在转录延伸过程中，新生RNA链从DNA模板上的置换效率很低。有趣的是，只有合成被置换RNA的聚合酶在5S rRNA基因终止子处终止；当新生RNA未从模板上被置换时，就会合成通读转录本。基于包括动力学特性、产生的精确3'末端、转录本从模板上的释放以及聚合酶的循环利用等多个标准，判断在5S rRNA基因终止子处具有3'末端的RNA是由真实的终止事件产生的。尽管最终只有40%的聚合酶分子在串联的5S rRNA基因终止子中的任何一个处终止，但实际上所有聚合酶都会在那里暂停，这表明终止信号识别在实验中可以与聚合酶释放相分离。因此，终止依赖于转录延伸过程中的RNA链置换，而终止信号识别则不然。我们根据转录终止的两步模型来解释我们的结果，其中聚合酶释放依赖于转录延伸过程中新生RNA链的命运。
