A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNA.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3 '-terminal CCA sequence that includes the site of aminoacylation, the additional 5 '-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3 '-CCA and 5 '-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNA(His) guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNA(His), which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNA(His) transcripts independent of the presence of G-1; however, tRNA(His) guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3 '-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.