Eukaryotic tRNA nucleotidyltransferase: synthesis, sorting and mechanism

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Transfer RNAs (tRNAs) are adaptor molecules that play an essential role in the conversion of information from DNA (its storage form) to protein (its functioning form). In organisms like us tRNAs do not work without the help of the enzyme tRNA nucleotidyltransferase which assists in their production. This enzyme functions in every location (cytosol, mitochondria, plastids) in a cell where tRNAs are needed. If we lack tRNA nucleotidyltransferase, we lack functional tRNAs, we can not make proteins and we die. Given the importance of this enzyme, we must answer some important questions. For example, how does a single gene residing in the nucleus code for multiple forms of this enzyme required in the cytosol, mitochondria and plastids? What are the signals on the enzyme that tell the cell where to send it and what factors in the cell recognize these signals? To answer these questions we will use mutagenesis to make altered forms of the enzyme (having or lacking potential targeting signals) and follow where they go in cells by linking them to easily detectable reporter proteins. The yeast and plant tRNA nucleotidyltransferases are much larger (at least one-third bigger) than their bacterial (E. coli) equivalent. Why are they bigger and can we target antifungal agents or herbicides to these extra regions? To explore this we are making changes in regions unique to the larger tRNA nucleotidyltransferases to see if these altered enzymes still support the life of yeast or, when purified, have catalytic activity in a test tube. We will use enzymatic and biophysical techniques to see how these changes alter enzyme structure and function. Finally, we made a mutant form of this enzyme that is extremely sensitive to elevated temperature and we found second site "suppressor" mutations that restore activity. What can the interactions between the mutant residues tell us about the structure and function of this important enzyme and how temperature affects it?