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

摘要

Nearly all tRNA^His 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 tRNA^His 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 tRNA^His 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 tRNA^His 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 tRNA^His A73 is overexpressed, and distinct, but milder growth defects, if tRNA^His C73 is overexpressed, these results show that the tRNA^His 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 tRNA^His C73, but not on tRNA^His 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 tRNA^His with A73, rather than C73, to prevent the possibility of 3′–5′ polymerization.