DNA-directed termination of RNA Polymerase II transcription II

RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.RNA聚合酶II（RNA polymerase II，RNAPII）的转录过程涵盖从启动子处起始转录、沿基因本体延伸转录产物，以及在下游终止子区域终止转录。与细菌不同，细菌的转录终止子通常包含特定DNA元件以引导RNAP解离¹，而学界此前认为RNAPII的转录终止完全由蛋白质辅助因子驱动¹⁻³。本研究借助生化重构实验，为RNAPII的转录终止机制提供了全新视角。出乎意料的是，仅由纯RNAPII介导的转录穿过终止子区域时，会在包含T串（T-tracts）的特定序列处发生显著程度的内在聚合酶解离现象。结合生物化学与单分子分析手段的结果表明，这类内在终止过程涉及RNAPII自发从DNA模板解离前的暂停，但未发生回溯。值得注意的是，尽管“鱼雷”模型中的Rat1-Rai1 RNA外切酶（人类中为XRN2）对暂停或停滞的聚合酶作用效率较低，但其可极大地促进内在终止过程。与之相反，延伸因子Spt4-Spt5（人类中为DSIF）则会抑制这类终止事件。在酵母中开展的全基因组3'端测序分析进一步佐证了该假说：转录终止发生于polyA位点处的转录本切割，该切割暴露出新的RNA末端，从而允许Rat1-Rai1鱼雷型外切酶的加载，随后该外切酶会追上在包含T串的内在终止位点处发生不稳定的RNAPII，最终完成转录终止。