Shundrovsky Alla, Santangelo Thomas J, Roberts Jeffrey W, Wang Michelle D
Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York, USA.
Biophys J. 2004 Dec;87(6):3945-53. doi: 10.1529/biophysj.104.044081. Epub 2004 Oct 1.
We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than approximately 100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to approximately 5 bp by using runoff transcription as an unambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DeltatR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or "backtracks," at the DeltatR2 pause sequence.
我们展示了一种技术，该技术允许使用单分子光镊技术对转录延伸进行序列依赖性分析。对单个RNA聚合酶（RNAP）分子的观察能够确定延伸动力学，而从大量研究中很难或无法准确获得这些动力学，并且能够在校准的机械负载下提供RNAP运动的高时间分辨率。先前单分子研究的一个局限性在于，难以将观察到的RNAP运动与其在DNA模板上的实际位置精确关联至优于约100个碱基对。在这项工作中，我们通过使用径流转录作为RNAP模板位置的明确标记，将转录光镊研究的空间精度提高到了约5个碱基对。这种径流方法足以明确地定位和研究单个已知的暂停序列（DeltatR2）。通过施加各种负载来辅助RNAP向前移位，我们专门研究了该暂停区域内的延伸动力学，发现暂停序列处的停留时间随着辅助负载的增加而减少。这一观察结果与大量生化研究一致，这些研究表明RNAP在DeltatR2暂停序列处会反向移位，即“回溯”。
