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

Allegheny College, Meadville PA

Investigators

Abstract

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.