Revyakin Andrey, Ebright Richard H, Strick Terence R
Howard Hughes Medical Institute, Waksman Institute, and Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway NJ 08854, USA.
Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4776-80. doi: 10.1073/pnas.0307241101. Epub 2004 Mar 22.
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 approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-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.
通过监测机械拉伸的超螺旋单链DNA分子的端到端延伸，我们能够直接观察到与RNA聚合酶（RNAP）解开约一圈启动子DNA相关的延伸变化。通过对负超螺旋和正超螺旋DNA进行平行实验，我们能够区分由RNAP依赖的DNA解旋引起的延伸变化（分辨率约为1个碱基对）和由RNAP依赖的DNA压缩引起的延伸变化（分辨率约为5纳米）。我们利用这种方法来量化解旋和压缩的程度、解旋和压缩的动力学，以及超螺旋、序列、ppGpp和核苷酸的影响。我们还利用这种方法检测连续的RNAP分子对启动子的清除和启动子的循环利用。我们发现，解旋复合物的形成速率和稳定性在很大程度上取决于超螺旋，并且超螺旋是通过机械方式（通过扭矩）而非结构方式（通过超螺旋的数量和位置）发挥作用的。该方法应该能够用于分析其他导致DNA扭曲和/或DNA压缩变化的核酸加工因子。
