Research Bio
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.
2013 Apr 10
Grant TD, Luft JR, Wolfley JR, Snell ME, Tsuruta H, Corretore S, Quartley E, Phizicky EM, Grayhack EJ, Snell EH. "The Structure of Yeast Glutaminyl-tRNA Synthetase and Modeling of Its Interaction with tRNA." Journal of molecular biology. 2013 Apr 10; Epub 2013 Apr 10. |
2012 Apr
Grant TD, Snell EH, Luft JR, Quartley E, Corretore S, Wolfley JR, Elizabeth Snell M, Hadd A, Perona JJ, Phizicky EM, Grayhack EJ. "Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase." Nucleic acids research. 2012 Apr 0; 40(8):3723-31. Epub 2011 Dec 17. |
2012
Dean KM, Grayhack EJ. "RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay." RNA (New York, N.Y.). 2012 18(12):2335-44. Epub 2012 Oct 24. |
2010
Letzring DP, Dean KM, Grayhack EJ. "Control of translation efficiency in yeast by codon-anticodon interactions." RNA (New York, N.Y.). 2010 16(12):2516-28. Epub 2010 Oct 22. |
2009 Sep
Quartley E, Alexandrov A, Mikucki M, Buckner FS, Hol WG, Detitta GT, Phizicky EM, Grayhack EJ. "Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding." Journal of structural and functional genomics. 2009 Sep 0; 10(3):233-47. Epub 2009 Aug 22. |
