CAREER: Molecular Interactions and Nucleotide Exchange Mechanism of Translation Factor EF1B Alpha

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9983565KinzyG-proteins are a broad class of proteins whose activity is regulated by the phosphorylation state of their bound nucleotide. When bound to the tri-phosphate form of guanosine (GTP), they are active in a multitude of biological processes such as protein synthesis. Stimulation of the G-protein's hydrolytic activity by another factor, a GTPase activating protein or GAP, converts the G-protein to the inactive diphosphate (GDP)-bound form. Another set of regulatory proteins termed guanine nucleotide exchange factors (GEFs) control the nucleotide bound state of G-proteins by facilitating the conversion back to the active GTP bound form. The translation Elongation Factor 1Balpha (eEF1Balpha, formerly EF-1beta) of the Baker's yeast Saccharomyces cerevisiae is a GEF for the elongation step of protein synthesis. As a small, essential protein, it serves as an excellent system to utilize biochemical and genetic approaches to the study of the mechanism of guanine nucleotide exchange and the regulation of the elongation phase of protein synthesis. eEF1Balpha functions on the G-protein eEF1A (formerly EF-1alpha) which is the homolog of the first G-protein for which the crystal structure was know, bacterial EF-Tu. eEF1A is active to bind and deliver activated amino acids attached to transfer RNAs to the ribosome only when in the GTP-bound form. The GDP to GTP exchange catalyzed by eEF1Balpha is essential to maintain a pool of active eEF1A and is likely the rate-limiting step of translation elongation. The PI's large bank of mutant forms of yeast eEF1Balpha has demonstrated the critical role of this protein in efficient and accurate protein synthesis in cells. The mechanism and regulation of the guanine nucleotide exchange process will be determined utilizing our available as well as new structurally based point mutations. Purified factors and in vitro assays for protein synthesis will identify the critical functional motifs of eEF1Balpha and dissect the interaction between this GEF and its G-protein eEF1A. The cellular growth and translation defects of yeast strain with a mutant form of eEF1Balpha allow us to utilize genetic approaches to identify factors that may regulate the GEF activity of this protein as well as the translation elongation process in general. Thus, the yeast S. cerevisiae is an eukaryotic system that permits simultaneous analysis of the role of eEF1Balpha in the translation elongation cycle in vivo and in vitro. The educational goals of this proposal utilize both existing minority enhancement programs and the work study program for undergraduates at Rutgers University in our research program to understand the role of eEF1Balpha in gene expression. The University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School (formerly Rutgers Medical School) has a tradition of supporting undergraduate research and minority enhancement programs. The work study placement program augments established programs with a system to identify economically disadvantaged students for laboratory research. The P.I., as the director of the UMDNJ RWJMS DNA Sequencing core facility, is also involved in the integration of state of the art molecular biology techniques into high school student and teacher programs and undergraduate courses at Rutgers University.