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

Abstract

Nearly all tRNAHis species have an additional 59 guanine nucleotide (G À1 ). G À1 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 À1 conservation suggest that G À1 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 39-59 (reverse) polymerization in vitro at the 59 end of activated tRNAs. Here we show that thg1-D 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 À1 addition activity. Since these thg1-D 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 À1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 39-59 polymerization in vivo on tRNA His C 73 , but not on tRNA His A 73 , demonstrating that the 39-59 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 39-59 polymerization.

Life without post-transcriptional addition of G−1: two alternatives for tRNAHis identity in Eukarya

“…The reason for eukaryotic Thg1 enzymes to have even retained the ability to recognize a Watson-Crick base pair was not understood. Previous studies that bypassed the essential requirement for Thg1 by overexpression of HisRS and tRNA His were complicated by the fact that there was still a strong growth defect evident in this strain, and whether this was due to incomplete aminoacylation or other defects associated with loss of Thg1 could not be completely decoupled (Preston and Phizicky 2010). However, the AcaHisRS-complemented thg1Δ strain grows similarly to the THG1-expressing strain under these conditions, and therefore will allow investigation of alternative function(s) of Thg1 enzymes, including those that may involve 3 ′ -5 ′ polymerase activity.…”

“…In S. cerevisiae Thg1 is essential for normal growth, due to the strong dependence of SceHisRS in the presence of the G −1 residue for tRNA His recognition. The requirement for Thg1 for viability can only be bypassed by overexpression of both SceHisRS and its tRNA His substrate, although these strains still exhibit a growth defect compared with THG1-containing yeast strains (Preston and Phizicky 2010). This suggests an epistasis between Thg1 function and tRNA His aminoacylation, and that the dependence on Thg1 could be bypassed by expression of a G −1 -independent HisRS in S. cerevisiae.…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…(Gu et al 2003). G 21 addition to tRNA His is essential for its aminoacylation (Gu et al 2005;Preston and Phizicky 2010 In humans usually only a subset of genes encoding a given isoacceptor contain an intron (http://lowelab.ucsc.edu/GtRNAdb/). In contrast, for S. cerevisiae and other fungi, generally all or the majority of duplicated copies of genes encoding a given tRNA isoacceptor will or will not contain an intron.…”

Doing it in reverse: 3′-to-5′ polymerization by the Thg1 superfamily

“…Thus, yeast Thg1 catalyzes a second biochemical activity, polymerizing Watson-Crick basepaired nucleotides in the opposite direction (39-to-59) to all studied DNA and RNA polymerases: the first example of an enzyme able to carry out this activity. Thg1-catalyzed 39-to-59 polymerization has also been observed in vivo in yeast using tRNA His variant substrates (Preston and Phizicky 2010), but a physiological function for the polymerization reaction in yeast remains unknown. Nonetheless, the unexpected ability of Thg1 to recognize and use Watson-Crick base pairs suggests the possibility of a biological function for the reverse polymerase activity.…”