The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase

Abstract

Nearly all tRNAHis species have an additional 5′ guanine nucleotide (G−1). G−1 is encoded opposite C73 in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNAHis guanylyltransferase (Thg1) opposite A73 in eukaryotes, and opposite C73 in other archaea. These divergent mechanisms of G−1 conservation suggest that G−1 might have an important cellular role, distinct from its role in tRNAHis charging. Thg1 is also highly conserved and is essential in the yeast Saccharomyces cerevisiae. However, the essential roles of Thg1 are unclear since Thg1 also interacts with Orc2 of the origin recognition complex, is implicated in the cell cycle, and catalyzes an unusual template-dependent 3′–5′ (reverse) polymerization in vitro at the 5′ end of activated tRNAs. Here we show that thg1-Δ strains are viable, but only if histidyl-tRNA synthetase and tRNAHis are overproduced, demonstrating that the only essential role of Thg1 is its G−1 addition activity. Since these thg1-Δ strains have severe growth defects if cytoplasmic tRNAHis A73 is overexpressed, and distinct, but milder growth defects, if tRNAHis C73 is overexpressed, these results show that the tRNAHis G−1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 3′–5′ polymerization in vivo on tRNAHis C73, but not on tRNAHis A73, demonstrating that the 3′–5′ polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNAHis with A73, rather than C73, to prevent the possibility of 3′–5′ polymerization.