Terri Goss Kinzy
Robert Wood Johnson Medical School
Dept of Biochemistry & Molecular Biology
Piscataway, NJ 08854
Regulation and mechanism of gene expression and protein synthesis and links to the cytoskeleton, infectious disease targets in translation
We are applying complementary genetic. Molecular, biochemical and structural techniques to dissect the mechanism of events occurring during protein synthesis. These include probing the physical and functional interaction of Elongations Factors (eEFs) with other factors that regulate gene expression. and the interaction between the G-proteins in elongation with the ribosome. The eEF1 protein complex is prototypical of all G-proteins. such as the oncogene Ras. and as such is regulated by a classic "GTPase" switch mechanism. The GTP-dependent activity of eEF1A is to deliver aminoacyl-tRNA to the ribosome and sense the accuracy of this process. The guanine nucleotide exchange factor (GEF) eEF1Balpha is essential in yeast and responsible for catalyzing the exchange of GDP for GTP to maintain the pool of active protein. Using a genetic system devoid of the eEF1Balpha protein allows us to manipulate eEF1A without its GEF to understand the regulation of G-protein activity and mutant forms of eEF1Balpha allow us to dissect the mechanism of guanine nucleotide exchange in vitro and the consequences of changes in this protein's activity in vivo. Lastly. the eEF1Bgamma subunit affects the sensitivity of the cell to oxidative stress and vacuolar function. Current work is addressing the implications of this finding in post-transcriptional control. In addition, eEF1A is an actin binding and bundling protein. Our work now shows an integrated model is emerging for organizing and regulating proteins synthesis in vivo. This actin binding is separate from the elongation activity and a target for 2 drugs we are studying for multiple medical applications.
Integrating an analysis of the two other factors involved in elongation, the translocase for the growing peptide chain eEF2 and the fungal specific factor eEF3 allows us to fully dissect the elongation cycle and to better understand their potential as drug targets. eEF2 is the target for diphtheria and pseudomonas toxins, and our work is addressing how these toxins attach the host cell. In addition, new work is addressing the association of eEF3 and the ribosome to determine how this interaction can be used for antifungal development. Using mutants as well as eEF3 from pathogenic fungal species and our structural data we are working towards developing targets for new drugs against this fungal specific essential proteins.