Structure and function of yeast tRNA ligase

Citation

Westaway, Shawn Kathleen (1993) Structure and function of yeast tRNA ligase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fasc-j790. https://resolver.caltech.edu/CaltechTHESIS:01102013-143442193

Abstract

The gene for yeast tRNA ligase has been sequenced and its transcription start sites have been mapped. Three other open reading frames in the vicinity of the tRNA ligase gene were characterized. One open reading frame, ORF4, is the yeast ARG3 gene. ORF1 is probably not transcribed or translated in yeast. ORF2 is an unidentified but essential gene in yeast.

A deletion of the central 200 amino acids has been engineered in the ligase protein. This deletion protein, designated DAC, was characterized in the in vitro tRNA splicing reaction with regard to the structure of the joined tRNA product. Cofactor requirements for tRNA joining activity and polynucleotide kinase activity were also determined. DAC possesses a GTP-dependent joining activity that is not manifested by wild-type ligase. In addition, both the wild-type and DAC proteins exhibit polynucleotide kinase activities that are more efficient with GTP than with ATP. Joining reactions with wild-type ligase indicate that joining of tRNA halves is more efficient in the presence of both GTP and ATP than with either cofactor alone. Wild-type tRNA ligase can incorporate they-phosphate of GTP into the splice junction of joined tRNA, but only when ATP is also provided. The ligase protein contains two distinct nucleotide triphosphate binding sites- one specific for GTP and one specific for ATP. A revised mechanism for tRNA splicing in yeast is presented.

Thesis Files