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：研究外围结构域对Dead-box蛋白Rok1酶功能的影响。核糖核酸在各种细胞过程中发挥关键作用。为了使RNAs在细胞中执行其特定的功能，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，这些变化对蛋白质加工功能非常重要。这项研究项目将开发基于化学的方法来识别和表征能够调节必要的RNA加工蛋白质的活性和初始结构状态的蛋白质结构元件。本研究的第二个目标是为对RNA-蛋白质研究感兴趣的学生提供一个有效的培训环境。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。为培养丰富的学习环境，将采用综合性多途径辅导和适时教学的实验研究。这个指导基础设施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，它在过去取得了成功。因此，该项目不仅将加深对蛋白质调控的理解，也将为科学领域的学生提供坚实的基础。DEAD-box蛋白构成了许多RNA代谢途径中必不可少的一大类推定解风酶。例如，核糖体RNA(RRNA)的加工和组装是高度调控的途径，需要几个死盒蛋白。这一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围元件/结构域来调节的。这项研究的目的是了解外周结构域的结构和热力学性质对模型蛋白质的活性和构象状态的影响。具体地说，将对Dead-box蛋白质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结果。将开发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区域和动态区域。由于模型蛋白及其人类同源物的解除调控具有关键的细胞后果，拟议的研究将进一步了解(1)DEAD-box蛋白如何在加工早期结合并在以后的事件中发挥作用，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，这些状态的特征对于任何利用动态多因素复合体中的ATP水解的蛋白质来说都是重要的。

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.