New Insights into the Mechanism of Initial Transcription

Background: RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts. Results: A mutant that aborts less during initial transcription releases the promoter at longer RNA lengths. Conclusion: Hybrid growth remodels the protein to disrupt promoter contacts; the protein, in turn, pushes back on the hybrid, leading to abortive instability. Significance: The model extends to multisubunit RNA polymerases. RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

背景：RNA聚合酶必须将核苷酸添加的能量学与启动子接触的定时释放相耦合。 结果：在初始转录过程中流产较少的一个突变体在更长的RNA长度时释放启动子。 结论：杂合链生长重塑蛋白质以破坏启动子接触；反过来，蛋白质对杂合链产生反作用，导致流产不稳定性。 意义：该模型扩展到多亚基RNA聚合酶。RNA聚合酶在从序列特异性的初始转录转变为稳定且相对序列非依赖性的延伸过程中经历大量结构和功能变化。最初，转录复合物通常不稳定，在延伸路径上产生短的流产产物。然而，在RNA聚合酶中已分离出能显著降低流产不稳定性的蛋白质突变。理解这些突变对于理解初始转录和启动子清除的能量学至关重要。我们在此证明，T7 RNA聚合酶中的P266L点突变，其流产循环显著减少，也更晚地过渡到延伸阶段，即在更长的RNA长度时。该突变体的这两个特性不一定相关联，相反，我们提出它们都源于对RNA - DNA杂合链驱动的启动子结合N末端平台旋转的屏障减弱，这种运动对于在向延伸转变过程中实现启动子接触的程序性定时释放是必要的。还讨论了多亚基RNA聚合酶中的类似情况。