RUI: Catalytic regulation of ribosome processing factors: Investigation of peripheral domain effects on the enzymatic capabilities of the DEAD-box protein Rok1p.

标题：RUI：核糖体加工因子的催化调节：对DEAD-box蛋白Rok1p酶促能力的外周结构域影响的研究。核糖核酸（rna）在多种细胞过程中起着关键作用。为了使rna在细胞中发挥其特定的功能，它们通常需要被蛋白质因子处理。蛋白质加工通常与结构变化有关，结构变化对加工蛋白质的功能非常重要。该研究项目将开发基于化学的方法来识别和表征可以调节基本RNA加工蛋白的活性和初始结构状态的蛋白质结构元件。本研究的第二个目的是为对rna -蛋白研究感兴趣的学生提供一个有效的训练环境。本科生将完成所有研究，包括本研究中使用的所有rna和蛋白质的合成和纯化。为了营造一个丰富的学习环境，我们将采用综合的多方法指导和实验研究的即时教学。这种指导基础结构旨在提高实验设计和分析中的问题解决和批判性思维技能，并且在过去取得了成功。因此，该项目不仅将进一步加深对蛋白质调控的理解，而且为学生在科学方面提供坚实的基础。DEAD-box蛋白构成了在许多RNA代谢途径中必不可少的一大类假定的解窗酶。例如，核糖体RNA （rRNA）的加工和组装是高度调控的途径，需要几种DEAD-box蛋白。这个蛋白质家族将细胞功能与蛋白质结构变化结合在一起。在DEAD-box蛋白的一个子集中，活性是通过额外的外周元件/结构域介导的。本研究的目的是了解外周结构域对模型蛋白活性和构象状态的影响的结构和热力学性质。具体来说，将对DEAD-box蛋白模型进行生化表征和比较分析，以评估结构域缺失变体的催化效果。将发展平衡和动力学方法来研究初始构象对核苷酸结合的影响。最后，将为模型系统确定溶剂可及的动态区域。由于模型蛋白及其人类同源物的解除管制具有关键的细胞后果，因此拟议的研究将进一步了解(1)DEAD-box蛋白在加工早期如何结合并在后期事件中发挥作用，以及(2)DEAD-box蛋白和其他RNA折叠和加工辅助因子如何利用各种构象来调节活性。因此，这些状态的表征对于在动态多因子复合体中利用ATP水解的任何蛋白质都是重要的。

Title: RUI: Catalytic regulation of ribosome processing factors: Investigation of peripheral domain effects on the enzymatic capabilities of the DEAD-box protein Rok1p.Ribonucleic acids, RNAs, play a critical role in a variety of cellular processes. In order for RNAS to perform their specific functions in cells they often need to be processed by protein factors. Protein processing is generally associated with structural changes that are very important to the processing protein function. This research project will develop chemical-based methodologies to identify and characterize the protein structural elements that can regulate the activity and initial structural state of essential RNA processing proteins. The second objective of this research is to provide an effective training environment for students interested in RNA-protein research. Undergraduate students will perform all investigations, including the synthesis and purification of all RNAs and proteins utilized in this study. To cultivate a rich learning environment, comprehensive multi-approach mentoring and Just-in-Time teaching of experimental research will be used. This mentoring infrastructure is designed to advance problem solving and critical thinking skills in experimental design and analysis, and it has been successful in the past. Thus, this project will not only further the understanding of protein regulation, but also provide a strong foundation for students in the sciences. DEAD-box proteins constitute a large sub-class of putative unwindases essential in many RNA metabolic pathways. For example, Ribosomal RNA (rRNA) processing and assembly are highly regulated pathways requiring several DEAD-box proteins. This family of proteins couples cellular function to protein structural changes. In a subset of DEAD-box proteins, activity is mediated through additional peripheral elements/domains. The goal of this research is to understand the structural and thermodynamic nature of peripheral domain effects on the activity and conformational state of a model protein. Specifically, a DEAD-box protein model will be biochemically characterized and comparatively analyzed to evaluate the catalytic consequence of domain deletion variants. Equilibrium and kinetic methodologies will be developed to study the effects of initial conformations on nucleotide binding. Lastly, solvent-accessible and dynamic regions will be identified for the model system. Since the deregulation of the model protein and its human homolog has critical cellular consequences, the proposed research will further the understanding of (1) how DEAD-box proteins can bind early during processing and function in later events, and (2) how DEAD-box proteins and other RNA folding and processing accessory factors can utilize various conformations to regulate activity. Thus, the characterization of these states is important for any protein that utilizes ATP hydrolysis within a dynamic multi-factor complex.