tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Yeast cytoplasmic tRNAs have 25 biochemically distinct modifications, and the average tRNA has 13 modified residues. These modifications are highly conserved in eukaryotes, including humans, and many of the corresponding genes have been identified in this and other labs. It is known from previous work in the field that several modifications in and around the anticodon loop have crucial roles in various aspects of translation, while several other modifications remote from the anticodon loop have specific roles in tRNA folding and/or stability. However, the precise roles of many other individual modifications are only poorly understood, and are under investigation in our lab.
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Another major problem of interest to us is the nature of the substrate specificity of modification enzymes. Modification enzymes are highly specific for certain tRNA species and for specific residues within these tRNAs. This specificity can be crucial because failure of modification can lead to non-functional tRNA, and because inadvertent modification of the incorrect species can have deleterious effects on the cell. We are currently investigating the specificity of several enzymes in vivo and in vitro.
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Recent work in this lab and the lab of E. Grayhack has focused on establishing yeast as the eukaryote of choice for high throughput purification of yeast and other eukaryotic proteins for biochemical analysis and structural biology. Although expression in E. coli has been used for biochemical analysis and 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 establish yeast as an organism for high throughput cloning, expression and purification, we have developed several methods.
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