RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay.
RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay.
复制标题
使用自切割转录物测定分析 T7 RNA 聚合酶转录过程中的 RNA 折叠。
DOI：
10.1021/bi00109a016
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Hearst,JE
中科院分区：
文献类型：
--
作者：
Tyagarajan,K;Monforte,JA;Hearst,JE
摘要：我们利用一种自裂RNA分子（“锤头”）研究了T7 RNA聚合酶转录过程中产生的RNA的长度依赖性折叠。转录物延伸被阻止在定义的位置使用链终止核糖核苷三磷酸类似物，3'-脱氧核苷三磷酸。当新生转录物达到转录物“锤头”结构域从三元复合体中完全出现所需的最小长度时，“锤头”结构形成并自裂，产生截断产物。这个实验产生了一个RNA测序阶梯，它终止于可以切割的长度；将该测序梯与使用非切割控制模板生成的序列梯进行比较。我们已经证明，在三元复合物中，转录物在经过切割点之前必须合成13个核苷酸，而通过加热从复合物中释放的RNA可以在切割位点之外仅存在3个或更多核苷酸时进行切割。结果表明，T7 RNA聚合酶中的RNA在三元络合物中并不是没有空间相互作用的，直到离聚合位点至少10个碱基时才可用于结构形成。结果表明，在三元配合物中，RNA-DNA杂交的最大可能长度为10。讨论了与其他RNA聚合酶，特别是大肠杆菌RNA聚合酶比较结果的相关性。
Revised Manuscript Received August 27, 1991 abstract: We have used a self-cleaving RNA molecule (a “hammerhead”) to study the length-dependent folding ofRNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates. When the nascent transcript attains the minimum length required for the “hammerhead” domain of the transcript to fully emerge from the ternary complex, the “hammerhead” structure formsand self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencingladder is compared to that generated by using a noncleaving control template. We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complexwhereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of po-lymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.