Combined Optical Trapping and Fluorescence Resonance Energy Transfer

组合光学捕获和荧光共振能量转移

• 批准号: 7623203
• 负责人: Matthew Paul Gordon
• 金额: $ 5.01万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623203
• 关键词: Affect; DNA-Directed RNA Polymerase; Data; Disease; Drug resistance; Fluorescence Resonance Energy Transfer; Genetic; Genetic Transcription; Label; Lead; Learning; Measurement; Messenger RNA; Pathway interactions; RNA; Regulation; Research; Role; Site; Structure; System; Techniques; Transcription-Coupled Repair; hammerhead ribozyme; instrument; laser tweezer; optical traps; premature; prevent; single-molecule FRET

DESCRIPTION (provided by applicant): Our current research focuses on RNA polymerase (RNAP). RNAP is an important target of genetic regulation, because of its role in almost all genetic pathways. RNAP frequently undergoes site-specific pausing, which affects recruitment of regulatory factors, transcription coupled repair, and termination. However, the specific structural mechanism which underlies pausing is not well understood. We can detect pausing of RNAP during elongation using optical tweezers, and by combining this with single molecule fluorescence resonance energy transfer (smFRET), we will detect structural changes in RNAP as a result of pausing.) Our specific aims are: 1) Develop a system for combining OT and smFRET measurements. We will present data showing that we have already accomplished this aim. 2) Apply this instrument to the study of RNA tertiary structure. Specifically, we plan to validate the instrument by using combined OT and FRET to study the how the tertiary contacts of the hammerhead ribozyme lead to changes in its structure and function. And, 3) Learn about the mechanism of RNAP pausing by observing the coordination of pausing with structural changes of the molecule. We will collaborate with Robert Landick to produce FRET-labeled RNA polymerase molecules suitable for FRET measurements, and use this, in conjunction with our instrument, to expand upon already existing Block-lab optical trapping techniques for studying RNAP pausing. RNAP pausing has a huge impact on regulation of RNAP transcription, and can lead to or prevent premature termination of mRNA transcription. Because of this, and because of RNAP's central role in genetic regulation, a better understanding of the mechanisms of RNAP pausing would have a significant impact on our understanding of genetic regulation as a whole, which in turn impacts almost every aspect of genetics, such as disease, drug resistance, and diversity.

描述（由申请人提供）：我们当前的研究重点是RNA聚合酶（RNAP）。 RNAP是遗传调节的重要目标，因为它在几乎所有遗传途径中的作用。 RNAP经常经历特定地点的暂停，这会影响调节因素，转录耦合修复和终止的募集。但是，尚未很好地理解了基础暂停的特定结构机制。我们可以使用光学镊子在伸长过程中检测RNAP的暂停，并通过将其与单分子荧光共振能传递（SMFRET）结合使用，我们将由于暂停而检测RNAP的结构变化。） 我们的具体目的是：1）开发一个用于组合OT和SMFRET测量的系统。我们将介绍数据，以表明我们已经实现了这一目标。 2）将此仪器应用于RNA三级结构的研究。具体而言，我们计划通过使用OT和FRET进行验证仪器，以研究Hammerhead核酶的三级接触如何导致其结构和功能变化。 3）通过观察与分子的结构变化的协调来了解RNAP暂停的机理。我们将与Robert Landick合作生产适合FRET测量的FRET标记的RNA聚合酶分子，并将其与我们的仪器结合使用，以扩展已经存在的块状LAB光学诱捕技术来研究RNAP暂停。 RNAP暂停对RNAP转录的调节有很大影响，并可能导致或防止MRNA转录的过早终止。因此，由于RNAP在遗传调节中的核心作用，对RNAP暂停机制的更好理解将对我们对整个遗传调节的理解产生重大影响，这反过来又影响了遗传学的各个方面，例如疾病，耐药性和多样性。