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Regulation of gene expression. mechanisms of protein synthesis and G-protein regulationThe goal of the work in our laboratory is to understand the structural and functional basis of G-protein regulation and post-transcriptional mechanisms that regulate gene expression. The components of the Translation Elongation apparatus in yeast. from soluble protein factors to the ribosome. allow an integrated approach to these questions. These components are targets for antibiotics and antifungals. mutant forms and inappropriate expression of these proteins are found in several human carcinomas. and mutations in several components affect the accuracy and efficiency of protein synthesis and viral replication. 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. Using this approach we have isolated a novel prion-like element in yeast. Lastly. the eEF1Bgamma subunit affects the sensitivity of the cell to oxidative stress. Current work is addressing the implications of this finding in post-transcriptional control using a proteomics approach. 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. We are currently performing X-ray crystallographic studies of the eEF1 complex. the eEF1A-actin interaction. and the other elongation factors eEF2 and eEF3. Additionally. we are part of an international consortium with groups from Denmark and U. Penn. using a combined genetic. biophysical and structural approach to the study of the yeast ribosome. Selected PublicationsOzturk SB, Kinzy TG. (2008) Guanine nucleotide exchange factor independence of the G-protein eEF1A through novel mutant forms and biochemical properties. J Biol Chem. Jun 18. [Epub ahead of print] Cai YC, So CK, Nie AY, Song Y, Yang GY, Wang LD, Zhao X, Kinzy TG, Yang CS. (2007) Characterization of genetic alteration patterns in human esophageal squamous cell carcinoma using selected microsatellite markers spanning multiple loci. Int J Oncol. 30(5):1059-67. Gross SR, Kinzy TG. (2007) Improper organization of the actin cytoskeleton affects protein synthesis at initiation. Mol Cell Biol. 27(5):1974-89. Anand M, Balar B, Ulloque R, Gross SR, Kinzy TG. (2006) Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A. J Biol Chem. 281(43):32318-26. Ozturk SB, Vishnu MR, Olarewaju O, Starita LM, Masison DC, Kinzy TG. (2006) Unique classes of mutations in the Saccharomyces cerevisiae G-protein translation elongation factor 1A suppress the requirement for guanine nucleotide exchange. Genetics. 174(2):651-63. Ortiz PA, Ulloque R, Kihara GK, Zheng H, Kinzy TG. Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance. J Biol Chem. (2006) 281(43):32639-48. Andersen CB, Becker T, Blau M, Anand M, Halic M, Balar B, Mielke T, Boesen T, Pedersen JS, Spahn CM, Kinzy TG, Andersen GR, Beckmann R. (2006) Structure of eEF3 and the mechanism of transfer RNA release from the E-site. Nature. 443(7112):663-8. Chatterjee I, Gross SR, Kinzy TG, Chen KY. (2006) Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression. Mol Genet Genomics. 275(3):264-76. Ortiz PA. Kinzy TG. (2005) Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast. Nucleic Acids Res. 33(18):5740-8. Anand. M.. Chakraburtty. K.. Marton. M.. Hinnebusch. A.. and Kinzy. T.G. (2003) Functional interactions between yeast translation eukaryotic elongation factors (eEF) 1A and eEF3. J. Biol. Chem. 278:6985-6991. Jørgensen. R.. Ortiz. P.A.. Carr-Schmid. A.. Nissen. P.. Kinzy. T.G. and Andersen. G.R. (2003) Two crystal structures demonstrate very large conformational changes of the eukaryotic ribosomal translocase. Nature Struct. Biol. 10:379-385. Kinzy. T.G.. Harger. J.W.. Carr-Schmid. A.. Kwon. J.. Shastry. M.. Justice. M. and Dinman. J.D. (2002) New targets for antivirals: the ribosomal A-site and the factors that interact with it. Virology 300: 60-70. Carr-Schmid. E.. Pfund. C.. Craig. E.A.. and Kinzy. T.G. (2002) Novel G-protein complex whose requirement is linked to the translational status of the cell. Mol. Cell. Biol. 22:2564-2574. Munshi. R.. Kandl. K.A.. Carr-Schmid. A.. Whitacre. J.L.. Adams. A.E.M.. and Kinzy. T.G. (2001) Overexpression of Translation Elongation Factor 1A affects the organization and function of the actin cytoskeleton in yeast. Genetics 157: 1425-1436. Andersen. G.R. Valente. L.. Pedersen. L.. Kinzy. T.G.. and Nyborg. J. (2001) Crystal structure of the eEF1A:eEF1Ba:GDP complex: an intermediate in nucleotide exchange. Nature Struct. Biol.8: 531-534. Andersen. G.R. Pedersen. L.. Valente. L.. Chatterjee. I.. Kinzy. T.G.. Kjeldegard. M. and Nyborg. J. (2000) Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Ba. Molecular Cell. 6: 1261-1266.
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