Abstract
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 tRNAHis 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 tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis 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.
Highlights
Transfer RNAs are essential key players in life
While in some Bacteria and Archaea the CCA sequence is encoded in the Transfer RNAs (tRNAs) genes, in all remaining organisms, especially in Eukarya, it is added post-transcriptionally by tRNA nucleotidyltransferase (CCA-adding enzyme) [2,3,4]
Knowing that tRNAHis as well as CCA-adding enzyme and Thg1 are localized in the cytosol of S. cerevisiae, we identified a sequential order of tRNAHis processing
Summary
Transfer RNAs (tRNAs) are essential key players in life. They function as adapter molecules, establishing an interface between the encoded genetic information in mRNA and the amino acid sequence at the protein level. The nucleotide binding pocket contains three highly conserved amino acids, forming Watson Crick-like hydrogen bonds to either CTP or ATP, without interacting with the tRNA [7] Despite these differences, both types of CCA-adding enzymes incorporate the nucleotides at high fidelity to the tRNA 3 -end. A precise dissociation constant for tRNAHis∆CCA was not attainable due to the inability to saturate the observed rate even at the highest concentration of protein achievable in our assays, and leading us to estimate that KD,app for tRNAHis∆CCA is ≥30 μM Taken together, these results clearly indicate that tRNAHis+CCA is a better substrate for Thg than tRNAHis∆CCA in terms of nucleotide incorporation rates, fidelity, and binding.
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