Investigation of RNA-Processing Protein Interactions and Dynamics with Simultaneous High-Resolution Tweezers and Fluorescence

RNA是细胞信息传递的中心，在将DNA编码的信息转化为蛋白质的过程中扮演中间人的角色。控制这种信息流的关键是RNA的产生和降解，这两个过程分别由利用能量的聚合酶蛋白质机器和外切体执行。第三种利用能量的RNA加工蛋白是解旋酶，它普遍存在，通过解压RNA双链来改变RNA的结构。一个新兴的主题是，RNA解旋酶是一种多功能机器，除了RNA解压外，还发挥着额外的作用，包括需要RNA锚定的能量和直接调节其他RNA加工蛋白质的活性。Ski2类解旋酶是一个新的解旋酶家族，它与聚合酶和外切体密切配合，影响广泛的RNA加工活动。这些解旋酶具有结合RNA双链传感和运动的新活性。该项目将研究这种新活性的基本分子机制，以揭示核心保守解旋酶成分的辅助修饰如何产生这些不同的功能。该项目还将研究这些新的解旋酶如何与其他RNA加工蛋白(例如解旋酶和聚合酶)合作和竞争。该项目还将利用密歇根州立大学开发的光学镊子和强大的科学推广活动来扩大现有的教育和推广计划，该计划基于与实验室内外单个细胞和RNA分子水平的生物物理科学实验的动手互动。该项目将通过开发和应用高分辨率、多模单分子技术来研究单一蛋白质和更大蛋白质机器组件的RNA加工蛋白质动力学。该项目将专门研究酵母Mtr4p解旋酶和相关的TraMP解旋酶+聚合酶系统，以及相关但功能不同的酵母死盒解旋酶Ded1p。MTR4P和TRAMP已经成为核RNA监测和处理的关键角色，这在一定程度上是通过精确修饰RNA以供外切体降解。尽管TRAMP只由三种蛋白质组成，但有两种是相反的极性分子机器--3‘到5’Mtr4p解旋酶和5‘到3’Trf4p聚合酶。Mtr4p具有多种功能，包括解开RNA，识别底物，调节聚合酶活性，以实现精确的RNA聚(A)标记。这些机器如何协调或竞争实现功能在很大程度上是有争议的，因为它们在RNA的5-10个核苷酸的规模上进行了高度动态的多自由度相互作用。该项目将能够解决有关MTR4P和TRAMP如何发挥作用的基本问题，使用前沿的单分子方法，同时结合高分辨率光学镊子和荧光。该项目将研究：(1)最近发现的Mtr4p的RNA双链传感易位机制是如何通过与突变体以及相关的Ded1p解旋酶进行比较来实现的，特别是在突变体中，关键结构域的丢失显著改变了活性。(2)mtr4p如何通过使用荧光和镊子同时直接测量解旋酶和聚合酶的活性来依次调节TRAMP聚合酶的活性。(3)相关的RNA解旋酶Ded1p解离机制是如何在与Mtr4p研究相同的机制分辨率下发生的。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，以应对新出现的对工具的需求，以研究超出单个分子规模的蛋白质-核酸相互作用，以及多蛋白质、多自由度复杂系统。该项目在密歇根州立大学提供了强有力的科学推广活动，以扩大现有的教育和推广计划，该计划基于与实验室内外单个细胞和RNA分子水平的生物物理科学实验的动手互动。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

RNA is central to cellular information transfer, acting as the intermediate in the transformation of DNA-encoded information into protein. Essential for controlling this information flow is both production and degradation of RNA, processes that are carried out by energy-utilizing polymerase protein machines and exosomes, respectively. A third type of energy-utilizing RNA processing proteins are helicases, which are ubiquitous and modify the structure of RNA by unzipping RNA duplexes. An emerging theme is that RNA helicases are multi-functional machines playing additional roles beyond RNA unzipping, including energy requiring RNA anchoring and directly regulating the activity of other RNA processing proteins. The Ski2-like helicases are a new family of helicases, which act in close coordination with polymerases and the exosome to affect a broad range of RNA processing activities. These helicases have novel activity coupling RNA duplex sensing and motion. The project will investigate the fundamental molecular mechanisms of this novel activity to reveal how these diverse functions can arise from the accessory modification of core, conserved helicase components. The project will also investigate how these novel helicases cooperate and compete with other RNA processing proteins, e.g., a helicase and polymerase. The project will also leverage the optical tweezers developed and strong science outreach activities at MSU to expand an existing education and outreach program, which is based on hands-on interactions with biophysical science experiments at the levels of individual cells and RNA molecules both within and out of the lab. 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. The project will specifically investigate the yeast Mtr4p helicase and the associated TRAMP helicase + polymerase system along with the related yet functionally distinct yeast DEAD-box helicase Ded1p. 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. While TRAMP is composed of only three proteins, two are opposing polarity molecular machines - the 3' to 5' Mtr4p helicase and the 5' to 3' Trf4p polymerase. Mtr4p performs multiple functions including unwinding RNA, discriminating substrates, and modulating polymerase activity to achieve precise poly(A) labeling of RNA. 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 project will be able to address the fundamental questions regarding how Mtr4p and TRAMP function using frontier single-molecule methods simultaneously combining high-resolution optical tweezers and fluorescence. The project will investigate: (1) How the recently discovered RNA-duplex-sensing translocation mechanism of Mtr4p is achieved via comparison to mutants, in particular one where the loss of a key domain dramatically alters activity, and to the related Ded1p helicase. (2) How the TRAMP polymerase activity is modulated in turn by Mtr4p via direct, simultaneous measurements of helicase and polymerase activity using fluorescence and tweezers. (3) How the related RNA helicase Ded1p unwinding mechanism occurs at the same mechanistic resolution as the Mtr4p studies. 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. The project provides strong science outreach activities at MSU to expand an existing education and outreach program, which is based on hands-on interactions with biophysical science experiments at the levels of individual cells and RNA molecules both within and out of the lab.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.