Confirmation of protein interactions involving tRNA nucleotidyltransferase from yeast two-hybrid data

A transfer RNA (tRNA) requires the nucleotide sequence cytidine, cytidine, adenosine at its 3 ' terminus to be functional. These nucleotides are added by the enzyme ATP (CTP): tRNA-specific tRNA nucleotidyltransferase (tRNA nucleotidyltransferase). In eukaryotes, this enzyme which is made in the cytosol must be targeted to specific subcellular destinations for tRNA maturation. Directing proteins to defined intracellular locations requires targeting signals on the protein of interest as well as other helper proteins. Previous studies have shown that AraYml079wp and AraGim1p, the products of the Arabidopsis Atlg19130 and Atlg29990 genes, respectively, interact with plant tRNA nucleotidyltransferases containing potential mitochondrial or chloroplast targeting signals. In this study, a heterologous E. coli expression system was developed to produce tRNA nucleotidyltransferase and the interacting proteins. Using these proteins a number of in vitro techniques including far Western blotting, co-elution and cross-linking were used to confirm the yeast two-hybrid data. In vitro results using co-elution and far Western blotting supported the in vivo results for an interaction between lupin tRNA nucleotidyltransferase and AraYml079wp. A number of experiments also were performed using a yeast YML079w deletion strain to try to elucidate a function for Yml079wp