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Associate Professor of Biochemistry and Biophysics
Ph.D. Cornell University 1981

Biochemical Genomics and Functional Proteomics

The linkage between a biochemical activity and the gene(s) that encode the responsible protein(s) reveals the chemical transformations undergone by small and large molecules and links those reactions to a particular biological process, frequently connecting biochemicals such as NAD with biology such as aging. To accelerate the association between genes and biochemical activities, we (myself, E. Phizicky [U. Rochester] and S. Fields [U. Washington]) produced a genomic array of purified yeast proteins (corresponding to known gene products) by cloning individual yeast genes fused to glutathione-S-transferase, and then purifying GST-ORF fusion proteins in pools. The gene responsible for a biochemical activity is identified from the entire proteome by assaying 64 pools of purified proteins for an activity, followed by deconvolution to a strain bearing a single GST-ORF fusion gene. More than 40 activities have been identified. To expand the biochemical and genetic utility of this approach as well as to include an active form of many membrane proteins and many newly recognized genes, a new array in which ORFs are fused at their C terminus to two purification tags is being constructed.

Cell-cycle regulated transcription

Cyclins play a key role in regulation of both growth and differentiation of the yeast S. cerevisiae. The CLB2 gene encodes an important cyclin involved in both passage through the G2/M phase of the cell cycle and in a nitrogen-regulated developmental decision at G₂/M between budding and pseudohyphal differentiation. Transcription of CLB2 is limited to the G₂/M phase of the cell cycle, is also regulated by nitrogen, by hypertonic stress and by positive feedback. While the proteins and mechanisms that regulate CLB2 transcription at G₂/M are known, the basis for the transcriptional response to nitrogen, stress or feedback is unknown. Our goal is to understand how these signals regulate transcription of CLB2. In particular, we have evidence that the essential Reb1 protein, previously unknown to have any role in CLB2 regulation, binds this promoter. Preliminary evidence indicates that a tRNA synthetase may transduce a signal for low nitrogen through the Reb1p bound at the CLB2 promoter.

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Recent Publications

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

Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S, Hol WG, Dumont ME, Phizicky EM, Grayhack EJ (2004) A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics, 3:934-8

Van Slyke C, Grayhack EJ (2003) The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region. Nucleic Acids Res, 31:4597-607

Phizicky EM, Martzen MR, McCraith SM, Spinelli SL, Xing F, Shull NP, Van Slyke C, Montagne RK, Torres FM, Fields S, Grayhack EJ (2002) Biochemical genomics approach to map activities to genes. Methods Enzymol, 350:546-59

Grayhack EJ, Phizicky EM (2001) Genomic analysis of biochemical function. Curr Opin Chem Biol, 5:34-9

Martzen MR, McCraith SM, Spinelli SL, Torres FM, Fields S, Grayhack EJ, Phizicky EM (1999) A biochemical genomics approach for identifying genes by the activity of their products. Science, 286:1153-5

Kuo MH, Nadeau ET, Grayhack EJ (1997) Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations. Mol Cell Biol, 17:819-32

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Contact Information

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