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首页> 外文期刊>Journal of Molecular Biology >Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability
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Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability

机译：停止的T7 RNA聚合酶延伸复合物的表征揭示了有助于稳定性的多种因素

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We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible "thumb" domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. Ln contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex. (C) 2000 Academic Press. [References: 54]
机译：我们已经构建了一系列质粒模板，这些模板允许T7 RNA聚合酶（RNAP）在启动子下游的固定间隔内在保留序列的情况下终止。虽然在+3至+6处停止的转录复合物非常不稳定，但在+10至+14处停止的复合物非常缓慢地解离，并逐渐失去扩展转录本的能力。停止在+18及以上的复合物更容易解离，但在存在下一个传入核苷酸的情况下，这些复合物的稳定性显着增强。出乎意料的是，发现超螺旋模板的复合物稳定性在+14或更高处停止，低于线性模板。为了进一步探索这一点，我们使用了合成DNA模板，其中非模板（NT）链的性质有所不同。起始复合物在互补NT链的存在下不稳定，而伸长复合物在互补NT链的存在下更加稳定，而非互补NT链（气泡错配）的存在则导致更大的稳定性。结果表明，NT链在置换新生RNA中起着重要作用，从而使其与RNAP中的RNA产物结合位点相互作用。 NT链也可以通过直接与酶相互作用而有助于稳定。在柔性“拇指”结构域中缺失的突变RNAP以与野生型（WT）酶相似的方式响应模板拓扑的变化，但是由突变酶形成的终止复合物特别依赖于NT链的存在以保持稳定性。相比之下，具有降低的结合单链RNA的能力的N端RNAP突变体形成的终止复合物具有比WT酶低得多的稳定性，并且这些复合物不会因NT链的存在而稳定。突变的RNAP对模板结构变化的不同响应表明，N末端和Thumb结构域在稳定转录复合物方面具有完全不同的功能。 （C）2000学术出版社。 [参考：54]

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