PDF(Pubmed)
Abstract：
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
摘要：
因子依赖性终止使用分子马达来重塑转录机制，但是相关的机制，尤其是在真核生物中，知之甚少。在这里，我们使用单分子荧光测定法来实时表征由Sen1解旋酶重塑的酿酒酵母转录终止复合物的组成和催化状态。我们确认Sen1以RNA转录本作为其底物，并通过水解多个ATPs来沿其易位，以形成具有停滞的RNA聚合酶II（PolII）转录延伸复合物（TEC）的中间体。我们表明，该中间体在水解单个ATP时解离，导致Sen1和RNA解离，之后，Sen1仍然与RNA结合。我们发现PolII最终处于多种状态：与DNA底物分离，这是通过转录气泡倒带而促进的，被保留在DNA底物上，或沿着DNA底物扩散。我们的结果为理解真核生物中Sen1依赖性转录终止的机制提供了完整的定量框架。
