Backtracking by single RNA polymerase molecules observed at near-base-pair resolution
Backtracking by single RNA polymerase molecules observed at near-base-pair resolution
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DOI：
10.1038/nature02191
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发表时间：
2003-12-11
期刊：
影响因子：
64.8
通讯作者：
Block, SM
中科院分区：
文献类型：
--
作者：
Shaevitz, JW;Abbondanzieri, EA;Block, SM
大肠杆菌RNA聚合酶（RNAP）在体内以显著的保真度合成RNA（1）。它的低错误率可以通过由两个连续事件组成的“校对”机制来实现。第一个事件（回溯）涉及RNAP通过几个碱基对的转录上游运动，其携带新生RNA转录物的30端远离酶活性位点。第二个事件（内切核酸裂解）发生后，一个可变的延迟，并在最近纳入的核糖核苷酸的断裂和释放，释放的活性位点的结果。在这里，通过将超稳定的光学捕获装置与一种新的双珠测定法相结合，以近碱基对的精度监测转录延伸，我们观察到RNAP单分子的回溯和恢复。回溯事件（类似于5 bp）很少发生在整个DNA模板的位置，并与暂停持续20秒至> 30分钟。肌苷三磷酸增加回溯暂停的频率，而辅助蛋白GreA和GreB，刺激新生RNA的切割，减少这种暂停的持续时间。
Escherichia coli RNA polymerase ( RNAP) synthesizes RNA with remarkable fidelity in vivo(1). Its low error rate may be achieved by means of a ' proofreading' mechanism comprised of two sequential events. The first event ( backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 30 end of the nascent RNA transcript away from the enzyme active site. The second event ( endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two- bead assay to monitor transcriptional elongation with near- base- pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (similar to 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to > 30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.