The requirement for the highly conserved G-1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase.
Nearly all tRNA(His) species have an additional 5' guanine nucleotide (G(-1)). G(-1) is encoded opposite C(73) in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNA(His) guanylyltransferase (Thg1) opposite A(73) in eukaryotes, and opposite C(73) 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-Delta 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-Delta strains have severe growth defects if cytoplasmic tRNA(His) A(73) is overexpressed, and distinct, but milder growth defects, if tRNA(His) C(73) 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) C(73), but not on tRNA(His) A(73), 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 A(73), rather than C(73), to prevent the possibility of 3'-5' polymerization.
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