Molodtsov Vadim, Anikin Michael, McAllister William T
Graduate Program in Cell and Molecular Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA; Department of Cell Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA.
Department of Cell Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA.
J Mol Biol. 2014 Sep 9;426(18):3095-3107. doi: 10.1016/j.jmb.2014.06.012. Epub 2014 Jun 27.
Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.
多亚基细菌RNA聚合酶（RNAP）的内在终止信号编码一个富含GC的茎环结构，其后是一个聚尿苷[poly(U)]序列，有人提出，茎环与RNAP出口孔的空间冲突会对下游RNA:DNA杂交体中的RNA施加剪切力，导致活性位点错位。结构不相关的T7 RNAP在类似类型的信号（TΦ）处终止，这表明存在一种共同的终止机制。在没有发夹结构的情况下（被动条件），T7 RNAP在同聚A和U序列中都能有效滑动，我们发现用易于滑动的A序列替换TΦ中的U序列仍能实现有效终止。在被动条件下，在聚（U）序列后掺入单个G残基（这是在TΦ处终止时的情况）会形成一个“锁定”复合物，该复合物无法延伸转录本。我们的结果支持这样一个模型，即下游存在易于滑动的序列会促进发夹与出口孔的空间冲突产生的剪切力的传递，从而导致活性位点发生改变，并形成一个锁定复合物，这代表了终止途径中的早期步骤。
