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）：我们目前的研究重点是RNA聚合酶（RNAP）。 RNAP 是遗传调控的重要靶点，因为它在几乎所有遗传途径中都发挥着作用。 RNAP 经常经历位点特异性暂停，这会影响调节因子的募集、转录偶联修复和终止。然而，暂停背后的具体结构机制尚不清楚。我们可以使用光镊检测延伸过程中 RNAP 的暂停，并将其与单分子荧光共振能量转移 (smFRET) 相结合，我们将检测由于暂停而导致的 RNAP 结构变化。） 我们的具体目标是： 1) 开发一个结合 OT 和 smFRET 测量的系统。我们将提供数据，表明我们已经实现了这一目标。 2）将该仪器应用于RNA三级结构的研究。具体来说，我们计划通过结合使用 OT 和 FRET 来验证仪器，以研究锤头核酶的三级接触如何导致其结构和功能的变化。并且，3) 通过观察暂停与分子结构变化的协调来了解 RNAP 暂停的机制。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，以扩展现有的用于研究 RNAP 暂停的 Block-lab 光学捕获技术。 RNAP暂停对RNAP转录的调节具有巨大影响，并且可以导致或阻止mRNA转录的提前终止。正因为如此，并且由于 RNAP 在遗传调控中的核心作用，更好地了解 RNAP 暂停机制将对我们对整个遗传调控的理解产生重大影响，进而影响遗传学的几乎每个方面，例如疾病、耐药性和多样性。