Joyce: Project overview

Joyce May-December 2009

Mapping the transcription start sites of Kluyveromyces lactis tRNA nucleotidyltransferase

Background

tRNA nucleotidyltransferase catalyzes the transfer of CCA to the 3' end of a tRNA molecule, allowing the subsequent step of aminoacylation by an aminoacyl-tRNA synthetase. tRNA nucleotidyltransferase is therefore an essential gene because it is required for aminoacylation and so translation.

In Saccharomyces cerevisiae, tRNA nucleotidyltransferase is targeted to the cytoplasm and the mitochondria. This targeting is achieved by the presence of three in-frame start codons (at amino acid positions 1, 10 and 18) which are initiated by three separate transcription start sites. Proteins generated from ATG1 produce variants that go to the mitochondrion, while proteins generated from ATG2 or ATG3 are targeted to the cytosol (and to a lesser degree, the nucleus). Removal of the first 9 or 17 amino acids didn't affect cytosolic targeting, but mitochondrial activity was reduced or lost; this implies that the first 9-17 amino acids play a significant role in mitrochondrial targeting.

NOTE TO SELF: ???This was determined by complementing a mutant of CCA1 with the ORF of either variant and testing for growth on glucose (tests for cytoplasmic tRNA nucleotidyltransferase activity) or glycerol (tests for mitochondrial activity)???. This is possible because a cell that is growing on glucose must be using glycolysis to survive, and since glycolysis is purely cytoplasmic, it must be able to generate the proteins needed for glycolysis in the cytoplasm, which means it must have been able to translated effectively in the cytoplasm with a fully functional tRNA nucleotidyltransferase. For glycerol, the cell needs to undergo oxidative phosphorylation in the mitochondrion, and if the cell can grow, it must have a functional tRNA nucleotidyltransferase that is present in the mitocondrion. /NOTE

In Kluyveromyces lactis, a yeast that is closely related evolutionary to S. cerevisiae, tRNA nucleotidyltransferase is also targeted to the mitochondrion and cytosol. K. lactis tRNA nucleotidyltransferase was isolated by generating a temperature-sensitive CCA1 mutant in S. cerevisiae and screening for temperature-resistant mutants by complementation with a K. lactis library. The identified sequence, KlCCA1, showed a high level of identity with S. cerevisiae CCA1 and supported growth on both glucose and glycerol, suggesting that this protein was capable of mitochondrial and cytosolic transport. K. lactis has two in-frame start codons (at amino acid positions 1 and 4) which are