Direct tests of the energetic basis of abortive cycling in transcription.
Direct tests of the energetic basis of abortive cycling in transcription.
复制标题
直接测试转录中失败循环的能量基础。
DOI：
10.1021/bi200620q
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Martin,CraigT
中科院分区：
文献类型：
--
作者：
Vahia,AnkitV;Martin,CraigT
尽管从DNA模板合成RNA通常是（并且必须是）非常稳定的过程以使得能够转录RNA酶转录物，但长期以来已知在最初转录期间，RNA复合物的前8-10个碱基相对不稳定，导致短的流产RNA转录物的释放。在过去的十年中，丰富的结构数据已经导致了具体的机制模型，阐述了早期的“强调中间”模型的初始转录。在这项研究中，我们测试这些模型中的每一个简单的模型酶T7 RNA聚合酶的基本预测。切口或缺口的非转录模板DNA的上游直接增长的杂交产生系统性的减少流产的下降，清楚地表明，压缩或“scrunching”的这种DNA是不是一个来源的功能不稳定性。类似地，在最初转录区域中的非模板链被错配或完全去除的DNA上的转录最多导致流产下降的适度减少，表明气泡的膨胀或“挤压”不是流产循环的主要驱动力。最后，从观察到的空间冲突的增长的杂交体对N-末端结构域的精力充沛的应力有助于最温和的流产循环，作为添加的空间散装（额外的RNA碱基）在上游端的杂交体不会导致预测的位置变化，在观察到的流产模式。我们的结论是，虽然结构的变化（scrunching）显然发生在初始转录，从这些变化的压力是不是主要的驱动力流产循环。
Although the synthesis of RNA from a DNA template is (and must be) a generally very stable process to enable transcription of kilobase transcripts, it has long been known that during initial transcription of the first 8–10 bases of RNA complexes are relatively unstable, leading to the release of short abortive RNA transcripts. A wealth of structural data in the past decade has led to specific mechanistic models elaborating an earlier “stressed intermediate” model for initial transcription. In this study, we test fundamental predictions of each of these models in the simple model enzyme T7 RNA polymerase. Nicking or gapping the nontranscribed template DNA immediately upstream of the growing hybrid yields no systematic reduction in abortive falloff, demonstrating clearly that compaction or “scrunching” of this DNA is not a source of functional instability. Similarly, transcription on DNA in which the nontemplate strand in the initially transcribed region is either mismatched or removed altogether leads to at most modest reductions in abortive falloff, indicating that expansion or “scrunching” of the bubble is not the primary driving force for abortive cycling. Finally, energetic stress derived from the observed steric clash of the growing hybrid against the N-terminal domain contributes at most mildly to abortive cycling, as the addition of steric bulk (additional RNA bases) at the upstream end of the hybrid does not lead to predicted positional shifts in observed abortive patterns. We conclude that while structural changes (scrunching) clearly occur in initial transcription, stress from these changes is not the primary force driving abortive cycling.
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影响因子：
5.6
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
I. Kuzmine;Craig T Martin
通讯作者：
Craig T Martin
影响因子：
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作者：
CARPOUSIS, AJ;GRALLA, JD
通讯作者：
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