Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation
Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation
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DOI：
10.1073/pnas.0307241101
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发表时间：
2004-04-06
影响因子：
11.1
通讯作者：
Strick, TR
中科院分区：
文献类型：
--
作者：
Revyakin, A;Ebright, RH;Strick, TR
通过监测机械拉伸的超螺旋单DNA分子的端到端延伸，我们已经能够直接观察到与RNA聚合酶(RNAP)解开大约一轮启动子DNA相关的延伸的变化。通过对负和正超螺旋DNA进行平行实验，我们已经能够解开依赖RNAP的DNA解卷引起的延伸变化(分辨率约为1-BP)和依赖RNAP的DNA压缩引起的延伸变化(分辨率约为5 nm)。我们已经使用这种方法来量化解卷和压实的程度，解卷和压实的动力学，以及超螺旋、序列、ppGpp和核苷酸的影响。我们还使用这种方法来检测连续的RNAP分子对启动子的清除和循环。我们发现，展开复合体的形成速度和稳定性在很大程度上取决于超卷，并且超卷的作用是机械的(通过扭矩)，而不是结构的(通过超级线圈的数量和位置)。该方法应允许分析引起DNA扭曲和/或DNA紧凑变化的其他核酸加工因素。
By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximate to1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximate to5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.