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tRNA Biosynthesis

One major project in the lab focuses on the biochemistry and biology of tRNA biosynthesis (together with Elizabeth Grayahck). tRNA processing is a surprisingly complex process, which involves numerous trimming, splicing, addition and modification steps of the initial tRNA transcript, as well as unexpectedly complicated intracellular trafficking and at least two quality control checkpoints. We are currently focusing on tRNA modifications. In the yeast Saccharomyces cerevisiae there are 25 biochemically distinct modifications that are found at 34 positions of the tRNA, and the average tRNA bears ~11 modifications. We have been interested in four questions concerning these modifications: First, what are the identities of the gene products that catalyze the modification reactions? To this end, we have used a biochemical genomics approach to assign a number of genes to different tRNA modification activities, and then confirmed these assignments in vivo. Second, what is the nature of the substrate specificity of these enzymes, and of the specificity for specific residues that are modified? This is important to understand because inadvertent modification of other RNAs or tRNAs, or modification of the incorrect sites, could have detrimental effects on cell growth. We are addressing this question by identification of the tRNA determinants that specify modification, and of the features of the protein that direct recognition of the tRNA substrates. Third, what are the catalytic properties of these proteins? Several of the proteins we have identified represent new classes of protein families and have interesting biochemical properties. In particular, we have recently shown that tRNA^His guanyltransferase, which normally adds a single guanine nucleotide to the 5' end of tRNA^His, can also polymerize RNA molecules in the reverse (3'-5') direction by addition of multiple nucleotides in a template-directed manner. Fourth, what is the biological role of tRNA modifications? This is important because the high degree of evolutionary conservation of many modifications suggests a crucial role for modifications, yet understanding of the roles of modifications has been elusive. Recent results demonstrate that modifications play a role in at least two pathways that monitor tRNA quality in the cell. Others have shown that lack of m¹A can lead to degradation of pre-tRNA_i^Met, by polyadenylation of tRNA by Trf4, followed by exonucleolytic degradation of the tRNA by the nuclear exosome. We have since shown that lack of m⁷G and m⁵C leads to degradation of pre-existing tRNA^Val(AAC) by another, as yet uncharacterized, pathway. We are currently working to understand the nature and scope of this pathway.

Engineering S. cerevisiae to produce proteins for x-ray crystallography (with Elizabeth Grayahck)

A second major project in the lab focuses on developing methodology for establishing yeast as the eukaryote of choice for structural biology, with a focus on small yeast protein complexes. Although expression in E. coli has been used for the determination of a large number of protein structures, expression of eukaryotic proteins in E. coli often results in limited solubility, as well as in the absence of post-translational modifications. To overcome these limitations, we are developing methods to enhance expression and purification of eukaryotic proteins in yeast. This project is part of the Center for High Throughput Structural Biology headed by George DeTitta at the Hauptman-Woodward Institute, and takes advantage of a comprehensive new library of yeast strains we recently constructed (the MORF library, constructed in collaboration with Mike Snyder at Yale and a group headed by Elizabeth Grayhack at Rochester), each expressing a yeast ORF from a plasmid under P_GAL control. Analysis of expression of each ORF demonstrates that at least a third of the strains produce high levels of fusion protein in S. cerevisiae (2 mg of protein per liter), and purification effected by affinity tags results in highly purified protein preparations. We are focusing on three problems to improve the versatility of yeast as a system for the preparation of proteins for structural biology. First, we are trying to modify yeast so that selenomethionine can be incorporated into proteins to facilitate obtaining phase information via multiwavelength anomalous dispersion. Incorporation of selenomethionine into proteins has been difficult in yeast because of the toxicity of the compound. Second, we are preparing new vectors to permit facile cloning (by ligation independent cloning methods) of multiple ORFs under inducible control. Third, we are developing methods for maximizing protein expression, for large scale purification of protein complexes in yeast.

A Systematic Screen for Over-expression Suppressors of Lethal Mutations

A third project in the lab focuses on the definition of functional modules in yeast, by systematic analysis of multi-copy suppression (together with Elizabeth Grayhack, Animesh Ray at the Keck Graduate Institute, and a group headed by David Galas at the Institute for Systems Biology in Seattle). In all organisms, complex cellular functions require multiple gene products that integrate into functional modules. Such modules, and the cross-talk between them, govern the most fundamental biological processes. While a conceptual framework of protein interactions can be constructed by analyzing individual gene products through biochemical and genetic approaches, the dynamics of intracellular networks--how modules interact and adapt according to cellular requirements—are often difficult to decipher. In an effort to better understand functional relationships among gene products, we are examining a specific type of genetic interaction- compensatory pairs, in which an otherwise lethal mutation is suppressed by over-expression of another gene(s). Although many such examples are known from the literature, a systematic analysis of a set of such compensated networks should provide insights on the robustness of functional networks in the context of network redundancies or functional modularity. We have begun a systematic search for over-expression suppressors of mutants by introducing a nearly complete library of ORFs under galactose regulation (the MORF library) into a series of mutant strains, and subsequently screening for suppression in the presence of galactose.