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

揭示生物分子复合物中的相关运动：Rho 解旋酶的转录终止

• 批准号: 80299817
• 负责人: Professor Dr. Philip Tinnefeld
• 金额: --
• 依托单位: Department Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/80299817?language=en
• 关键词: Revealing; correlated; movements; biomolecular; complexes

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.

解旋酶是一种广泛存在的分子马达酶，在许多重要的核酸交换过程中发挥重要作用。六聚体解旋酶Rho是一种转录终止因子，其结合新生RNA并将其从转录复合物中解离。这是本项目的目的是可视化的行动，单一的Rho解旋酶和剖析其工作机制，使用多色单分子光谱。单分子方法将能够在生物学相关条件下真实的实时监测特定的结构变化。扩展最近开发的可重复性能力，我们打算（i）定量测定生物分子复合物的化学计量，（ii）基于几个荧光团之间的荧光共振能量转移（FRET）的三维结构的确定，以及（iii）生物分子动力学的可视化，包括生物分子复合物内不同片段的相对运动。该方法将使用DNA连接，是有前途的候选人FRET为基础的荧光传感器进行评估。随后，我们将使用Rho，RNA和DNA上的荧光标记来研究Rho解旋酶活性的动力学。因此，可以回答重要的问题，例如Rho是否在易位后离开其初始结合位点。其愿景是解决单分子技术日益复杂的问题，如体外转录机制的重建。