RNA FOLDING DURING TRANSCRIPTION BY ESCHERICHIA-COLI RNA-POLYMERASE ANALYZED BY RNA SELF-CLEAVAGE
RNA FOLDING DURING TRANSCRIPTION BY ESCHERICHIA-COLI RNA-POLYMERASE ANALYZED BY RNA SELF-CLEAVAGE
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DOI：
10.1021/bi00486a015
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发表时间：
1990-08-28
期刊：
影响因子：
2.9
通讯作者：
HEARST, JE
中科院分区：
文献类型：
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作者：
MONFORTE, JA;KAHN, JD;HEARST, JE
我们使用了一个与植物类病毒亚序列(“锤头”)相关的自裂解RNA分子来研究大肠杆菌RNA聚合酶在转录过程中产生的RNA的长度依赖折叠。使用链终止的核糖核苷三磷酸类似物(3‘’-脱氧NTP‘’S或3‘’-O-甲基INTP‘’S)在特定的位置阻止转录本的延长。当转录本可以形成“锤头”结构时，它就会自我分解，得到截断的产物。该实验产生了终止于可能切割的长度的RNA测序梯形图；将该测序梯形图与使用非切割转录本或使用[α-硫代]三磷酸腺苷代替三磷酸腺苷所产生的梯形图进行比较。我们已经证明，超过裂解点的RNA必须合成15-18个核苷酸(NT)，然后转录本才能在三元复合体中自裂解，而通过加热从复合体中分离出来的RNA目前只有3个或更多的核苷酸在裂解点之外进行切割。既有序列依赖效应，也有长度依赖效应。结果表明，12.+-。1个NT被隔离在三元复合体中，这与其他人提出的转录泡中DNA-RNA杂交的存在是一致的。结果表明，“锤头”结构不会破坏杂交。杂交种之外的RNA似乎不受与酶的相互作用的限制，因为自裂解结构的最后一茎在组成它的RNA从DNA-RNA杂交物中出现时就形成了。转录本的自我切割为研究特性较差的转录复合体提供了一个简单的结构探针。讨论了结果与转录终止模型的相关性。
We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during trnascription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3''-deoxyNTP''s or 3''-O-methylINTP''s). When the transcript can form the "hammerhead" structure it self-calves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [.alpha.-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more at present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 .+-. 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.