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Associate Professor of Biochemistry and Biophysics
Ph.D. Cornell University 1981
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Functional Proteomics, Genomics, Gene Expression in the yeast S. cerevisiae
Functional Proteomics: The wealth of sequenced genomes has resulted in the development of powerful genomic approaches to define the functions of the individual genes, to deduce the networks of interactions between genes and to mine additional information encoded in the genome. For example, a collection of 5,854 sequence-verified yeast expression plasmids (constructed in collaboration with Eric Phizicky, Mark Dumont and Mike Snyder) has opened the door to study signals within genes that regulate their expression. We observe 1,000 fold differences in expression of genes in this collection, despite the fact that all ORFs are expressed using the same set of extragenic regulatory signals. Thus, the ORFs themselves or their protein products are esponsible for these large differences in expression. Analysis of this collection has led to the two projects described below.
The roles of codons in gene expression and regulation: Although accurate and efficient translation of mRNA into proteins is crucially important for the cell, not all of the 61 codons that specify insertion of amino acids into nascent polypeptides behave equally in this process. In fact, almost all synonymous codons, i.e. those that specify the same amino acid, are used at very different frequencies within most organisms, including the yeast S. cerevisiae Although codon use has been implicated as a determinant of expression levels, neither the identity, nor the defining characteristics of codons that impair expression are known. Studies in the Grayhack laboratory are designed to define the rules and mechanisms by which specific codons, and arrangements of codons, impair gene expression, and to identify regulatory models dictated by codon usage in the yeast genome.
Developing yeast to express protein complexes for structural analysis: The structures of many eukaryotic proteins and protein complexes are still unknown because many eukaryotic proteins are either not soluble in E. coli or bear post-translational modifications that are not added in E. coli. To this end we developed a general solution to incorporate selenomethionine into proteins expressed in this yeast, for use in MAD phasing. In addition, we have developed vectors for simultaneous high level expression and affinity purification of protein complexes from S. cerevisiae. Our current goal is to explore the limits of protein expression, solubility and folding to learn in part how yeast differs from E. coli, and what factors contribute most to gene expression.
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Visit my Lab Page
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Chernyakov I, Whipple JM, Kotelawala L, Grayhack EJ, Phizicky EM (2008) Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5'-3' exonucleases Rat1 and Xrn1. Genes Dev, 22:1369-80
Kotelawala L, Grayhack EJ, Phizicky EM (2008) Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species. Rna, 14:158-69
Jackman JE, Grayhack EJ, Phizicky EM (2008) The use of Saccharomyces cerevisiae proteomic libraries to identify RNA-modifying proteins. Methods Mol Biol, 488:383-93
Chernyakov I, Baker MA, Grayhack EJ, Phizicky EM (2008) Chapter 11. Identification and analysis of tRNAs that are degraded in Saccharomyces cerevisiae due to lack of modifications. Methods Enzymol, 449:221-37
Wilkinson ML, Crary SM, Jackman JE, Grayhack EJ, Phizicky EM (2007) The 2'-O-methyltransferase responsible for modification of yeast tRNA at position 4. Rna, 13:404-13
White MA, Clark KM, Grayhack EJ, Dumont ME (2007) Characteristics affecting expression and solubilization of yeast membrane proteins. J Mol Biol, 365:621-36
Malkowski MG, Quartley E, Friedman AE, Babulski J, Kon Y, Wolfley J, Said M, Luft JR, Phizicky EM, DeTitta GT, Grayhack EJ (2007) Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci U S A, 104:6678-83
Jackman JE, Kotelawala L, Grayhack EJ, Phizicky EM (2007) Identification and characterization of modification enzymes by biochemical analysis of the proteome. Methods Enzymol, 425:139-52
Phizicky EM, Grayhack EJ (2006) Proteome-scale analysis of biochemical activity. Crit Rev Biochem Mol Biol, 41:315-27
Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM (2006) Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell, 21:87-96
Gu W, Hurto RL, Hopper AK, Grayhack EJ, Phizicky EM (2005) Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C. Mol Cell Biol, 25:8191-201
Alexandrov A, Grayhack EJ, Phizicky EM (2005) tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p. Rna, 11:821-30
Ma X, Yang C, Alexandrov A, Grayhack EJ, Behm-Ansmant I, Yu YT (2005) Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism. Embo J, 24:2403-13
Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, Grayhack EJ (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev, 19:2816-26
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E-Mail: Elizabeth_Grayhack@urmc.rochester.edu
Elizabeth J. Grayhack
Department of Biochemistry and Biophysics
University of Rochester School of Medicine and Dentistry
601 Elmwood Ave, Box 712
Rochester, New York 14642
Office: Medical Center 3-7415
Telephone: (585) 275-2765; Fax: (585) 275-6007
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