Paused RNA polymerase II is the major regulated step of transcription in metazoans. We describe here a unique human cell-free transcription system that recapitulates RNA pol II pausing and assemble paused pol IIs on the human CMV IE, SV40, and heat shock promoters, all is the case in vivo. We then use the system to show that PARP-1 and CDK12/13 inhibitors directly affect pausing and elongation. We then show that O-GlcNAcylation is required for the establishment of a paused pol II: inhibition of OGT allowed pol II to bypass pausing and begin to elongate. Addback of rOGT or the pausing factor NELF re-established the pausing. In vivo nascent RNA measurements showed that OGA inhibition blocks elongation. These data show that cell-free systems can recapitulate RNA pol II pausing, that PARP-1 and CDK12/13 directly regulate RNA pol II elongation, and identify O-GlcNAc cycling regulating both pausing and pause release.

中文翻译：

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人RNA Pol II暂停系统的建立和调控Pol II暂停和延伸的O-GlcNAc循环的鉴定

暂停的RNA聚合酶II是后生动物转录的主要调控步骤。我们在这里描述了一个独特的人类无细胞转录系统，该系统概述了RNA pol II暂停并在人CMV IE，SV40和热休克启动子上组装了暂停的pol II，所有情况在体内都是如此。然后，我们使用该系统显示PARP-1和CDK12 / 13抑制剂直接影响暂停和延长。然后，我们显示O-GlcNAcylation是建立暂停的pol II所必需的：对OGT的抑制使pol II可以绕过暂停并开始延长。rOGT的加法或暂停因子NELF重新建立了暂停。体内新生RNA测量表明，OGA抑制作用会阻止延伸。这些数据表明，无细胞系统可以概括RNA pol II暂停，PARP-1和CDK12 / 13直接调节RNA pol II的延伸，