Investigation of RNA-Processing Protein Dynamics with Simultaneous High-Resolution Optical Traps and Single-Molecule Fluorescence

职务名称：同时使用高分辨率光阱和单分子荧光研究RNA加工蛋白质动力学在该项目中，PI将开发光学方法，以实时直接观察参与RNA加工的蛋白质机器的活动。 RNA是细胞信息的重要载体之一，其产生和降解受到与蛋白质机器相关的RNA蛋白质相互作用的严格控制。该项目的总体目标是研究不同分子机器之间的合作和竞争如何导致细胞中信息的仔细调节。该项目开发的技术将适用于RNA加工中涉及的蛋白质机器之外，包括各种各样的复杂蛋白质系统。该项目将利用光学方法的开发以及密歇根州立大学强大的艺术和科学联系，开发一项教育和外展计划，重点是在单个细胞和RNA分子水平上与生物物理科学进行实践互动。PI将为学生和公众创造一个视频游戏实验室体验。该项目将通过开发和应用高分辨率，多模态单分子技术来研究单个蛋白质和更大蛋白质机器组装体的RNA加工蛋白质动力学。具体而言，PI将研究酵母核RNA解旋酶Mtr 4p和相关TRAMP蛋白复合物。Mtr 4p和TRAMP已经成为核RNA监测和加工的关键参与者，部分通过精确修饰RNA以供外泌体降解。这些机器如何协调或竞争以实现功能在很大程度上是有争议的，因为它们在RNA的仅5-10个核苷酸的尺度上具有高度动态的多自由度相互作用。 PI将解决有关Mtr 4p和TRAMP如何使用单分子和高分辨率光镊和荧光发挥作用的基本问题。结合高分辨率光学捕获和多色荧光的先进技术将广泛应用于所有生物物理分子动力学研究，以满足对工具的新需求，从而研究蛋白质-核酸相互作用，超越单个分子尺度，走向多蛋白质，多自由度的复杂系统。

Title: Investigation of RNA-processing protein dynamics with simultaneous high-resolution optical traps and single-molecule fluorescenceIn this project, the PI will develop optical methods that will enable direct observation in real-time of the activity of protein machines that are involved in processing RNA. RNA is one of the essential carriers of cell information, and its production and degradation are tightly controlled by RNA protein interactions associated with protein machines. The overall goal of this project is to investigate how both the cooperation and competition between different molecular machines lead to the careful regulation of information in the cell. The techniques developed in this project will apply beyond the protein machines involved in RNA processing to include a broad assortment of complex protein systems. This project will leverage optical methods development and the strong art and science connection at Michigan State University to develop an education and outreach program centered on hands-on interactions with biophysical science at the individual cell and RNA molecule level. The PI will create a video game lab experience aimed at students and the public.This project will investigate RNA-processing protein dynamics of single proteins and larger protein machine assemblies by developing and applying high-resolution, multi-modal single molecule techniques. Specifically, the PI will investigate the yeast nuclear RNA helicase Mtr4p and the associated TRAMP protein complex. Mtr4p and TRAMP have emerged as key players in nuclear RNA surveillance and processing in part via precise modification of RNA for degradation by the exosome. How these machines coordinate or compete to achieve function is controversial in large part due to their highly dynamic, multi-degree of freedom interactions on the scale of only 5-10 nucleotides of RNA. The PI will address the fundamental questions regarding how Mtr4p and TRAMP function using single-molecule and high resolution optical tweezers and fluorescence. Advanced techniques combining high-resolution optical trapping and multi-color fluorescence will apply broadly across all biophysical molecular dynamics investigations towards the emerging need for tools to investigate protein-nucleic acid interactions beyond the individual molecule scale towards multi-protein, multi-degree of freedom complex systems.