Revealing correlated movements in biomolecular complexes: transcription termination by Rho helicase

解旋酶是一种广泛存在的分子马达酶，是许多重要的核酸交易所必需的。六聚体解旋酶Rho是一种转录终止因子，它与新生RNA结合，并将其从转录复合体中解离。本项目的目的是用多色单分子光谱学可视化单个Rho解旋酶的作用，并剖析其作用机制。单分子方法将能够实时监测生物相关条件下的特定结构变化。扩展了最近开发的多色能力，我们打算：(I)生物分子络合物化学计量的定量测定，(Ii)基于几个荧光团之间的荧光共振能量转移(FRET)来确定3D结构，以及(Iii)生物分子动力学的可视化，包括生物分子络合物中不同链段的相对运动。该方法将使用有望成为基于FRET的荧光传感器的候选DNA的DNA连接进行评估。随后，我们将利用Rho、RNA和DNA上的荧光标记来研究Rho解旋酶活性的动态。因此可以回答一些重要的问题，例如Rho在易位时是否离开了它的初始结合部位。我们的愿景是用单分子技术解决日益复杂的问题，例如体外转录机制的重建。

Helicases are widespread molecular motor enzymes required for numerous essential nucleic-acid transactions. The hexameric helicase Rho is a transcription termination factor that binds to the nascent RNA and dissociates it from the transcription complex. It is the aim of this project to visualize the action of single Rho helicases and to dissect their working mechanism using multi-color singlemolecule spectroscopy. The single-molecule approach will enable to monitor specific structural changes under biologically relevant conditions in real time. Expanding the recently developed multicolor capability we intend (i) the quantitative determination of stoichiometries of biomolecular complexes, (ii) the determination of 3D structures based on Fluorescence Resonance Energy Transfer (FRET) between several fluorophores, and (iii) the visualization of biomolecular dynamics including relative movements of different segments within biomolecular complexes. The methodology will be evaluated using DNA junctions that are promising candidates for FRET based fluorescent sensors. Subsequently we will investigate the dynamics of Rho helicase activity using fluorescent labels on Rho, RNA and DNA. Important questions e.g. whether Rho leaves its initial binding site upon translocation can thus be answered. The vision is to address problems of increasing complexity with single- molecule techniques such as the reconstitution of the transcription machinery in vitro.