Abstract:
Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3\' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3\' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
摘要:
RNA聚合酶II(RNAPolII)的转录终止通过切割和聚腺苷酸化因子(CPF或CPSF)与RNA3'末端加工连接。CPF含有核酸内切酶,聚(A)聚合酶,和蛋白磷酸酶活性,切割和聚腺苷酸化pre-mRNA和去磷酸化RNAPolII以控制转录。RNA3末端加工机制究竟是如何与转录偶联的,目前尚不清楚。这里,我们结合了体外重建,结构研究,和全基因组分析表明,酵母CPF在物理和功能上与RNAPolII相互作用。令人惊讶的是,CPF介导的去磷酸化在体外促进RNAPolII茎到茎同源二聚体的形成。该二聚体与转录相容,但与转录延伸因子的结合不相容。细胞中二聚化界面的破坏会导致转录缺陷,包括蛋白质编码基因上RNAPolII丰度的改变,tRNA基因,和基因间区域。我们假设RNAPolII二聚化可能为转录终止的变构模型提供机制基础。