Baumann Christoph G, Cross Stephen J
Department of Biology, University of York, York, UK.
Methods Mol Biol. 2011;778:175-91. doi: 10.1007/978-1-61779-261-8_12.
RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique "three-bead" geometry. This single-molecule transcription assay can be used to probe the mechanics of both stationary and moving RNAP-DNA complexes engaged in different stages of transcription.
RNA聚合酶（RNAP）是一种依赖DNA的马达蛋白，它通过其活性位点将核糖核苷酸聚合与力的产生和DNA易位联系起来，即机械功。使用光镊的单分子研究使研究人员能够探究参与转录延伸的单亚基病毒以及多亚基原核和真核RNA聚合酶的负载依赖性核糖核苷酸掺入率和持续合成能力。本文描述了一种单分子方法，该方法允许使用双阱光镊和独特的“三珠”几何结构实时跟踪完整的转录周期（即启动子结合、起始、延伸和终止）。这种单分子转录测定法可用于探究参与转录不同阶段的固定和移动RNAP-DNA复合物的力学特性。
