Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

Abstract

All histidine tRNA molecules have an extra nucleotide, G-1, at the 59 end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNA His , G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNA His identity. Specifically, the 59-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute ; ;4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNA His , were used to facilitate an atomic group ''mutagenesis'' study of the ÿ1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNA His for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 59 monophosphate of yeast tRNA His is critical for aminoacylation by yeast HisRS and contributes ; ;3 kcal/mol to transition state stability. The primary role of the unique ÿ1:73 base pair of yeast tRNA His appears to be to properly position the critical 59 monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNA His interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.

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

“…Furthermore, since the SUP4°suppressor is unable to suppress a kex2-H213oc mutant relative to its vector control (0.06% vs. 0.15%), we conclude that suppression of kex2-H213 nonsense mutants provides an effective test for histidine insertion at this catalytically important site. (Rudinger et al 1994;Nameki et al 1995;Gu et al 2005;Rosen et al 2006). In support of this explanation, we find that overproduction of Thg1 increases mating of strains overexpressing tRNA His am A 73 , from 0.01% to 5.5% (Table 1).…”

“…An essential G À1 addition activity of Thg1 is also consistent with biochemical evidence that the G À1 residue of tRNA His is a strong determinant for histidylation by Hts1 (Rudinger et al 1994;Nameki et al 1995;Gu et al 2005;Rosen et al 2006), and with the coincident deacylation of tRNA His and His was spotted on a silica thin-layer chromatography (TLC) plate, and resolved in solvent containing n-propanol: NH 4 OH:H 2 O (55:35:10) for about 16 h, dried, and radioactivity was visualized using a Storm PhosphorImager. Note: the migration of the solvent front was not uniform in this TLC experiment.…”

“…The predominant mechanism requires the G À1 residue or its phosphate as a crucial identity element for the corresponding histidyl-tRNA synthetase (HisRS), based on experiments in E. coli and yeast (Himeno et al 1989;Francklyn and Schimmel 1990;Rudinger et al 1994;Nameki et al 1995;Fromant et al 2000;Connolly et al 2004;Rosen and MusierForsyth 2004;Gu et al 2005;Rosen et al 2006). Since the G À1 residue is unique to tRNA His , there would be strong evolutionary pressure to maintain this recognition element once it had evolved.…”

“…Indeed, one welldocumented role of the G À1 residue is for aminoacylation of tRNA His . The G À1 residue or its phosphate is a strong identity element in vitro for histidylation of E. coli tRNA His and yeast tRNA His by their corresponding synthetases (Himeno et al 1989;Francklyn and Schimmel 1990;Rudinger et al 1994;Nameki et al 1995;Fromant et al 2000;Connolly et al 2004;Rosen and Musier-Forsyth 2004;Rosen et al 2006), and in vivo in yeast, based on the concomitant loss of G À1 from tRNA His and accumulation of deacylated tRNA His that occurs as Thg1 is depleted (Gu et al 2005). Thus, the G À1 residue may have been retained during evolution primarily as a determinant for histidyl-tRNA His synthetases.…”

tRNA His guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases

“…Thg1 catalyzes the highly unusual 3′-5′ addition of a single guanine to the 5′-end of tRNA His (1,2). This reaction is an obligatory step in the maturation of this tRNA because the extra 5′ base, G −1 , constitutes a primary identity element for the aminoacyl-tRNA synthetase (HisRS) that attaches the amino acid histidine to the 3′-end of the tRNA (3)(4)(5)(6)(7)(8)(9). Thg1 is thus essential for maintaining the fidelity of protein synthesis.…”