tRNA^His guanylyltransferase adds G₋₁ to the 5′ end of tRNA^His by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases

Abstract

All eukaryotic tRNAHis molecules are unique among tRNA species because they require addition of a guanine nucleotide at the ÿ1 position by tRNA His guanylyltransferase, encoded in yeast by THG1. This G ÿ1 residue is both necessary and sufficient for aminoacylation of tRNA by histidyl-tRNA synthetase in vitro and is required for aminoacylation in vivo. Although Thg1 is presumed to be highly specific for tRNAHis to prevent misacylation of tRNAs, the source of this specificity is unknown. We show here that Thg1 is >10,000-fold more selective for its cognate substrate tRNA His than for the noncognate substrate tRNAPhe . We also demonstrate that the GUG anticodon of tRNA His is a crucial Thg1 identity element, since alteration of this anticodon in tRNA His completely abrogates Thg1 activity, and the simple introduction of this GUG anticodon to any of three noncognate tRNAs results in significant Thg1 activity. For tRNA Phe , k cat /K M is improved by at least 200-fold. Thg1 is the only protein other than aminoacyl-tRNA synthetases that is known to use the anticodon as an identity element to discriminate among tRNA species while acting at a remote site on the tRNA, an unexpected link given the lack of any identifiable sequence similarity between these two families of proteins. Moreover, Thg1 and tRNA synthetases share two other features: They act in close proximity to one another at the top of the tRNA aminoacyl-acceptor stem, and the chemistry of their respective reactions is strikingly similar.

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

“…We find that His-tRNA His lacking the G À1 residue is nearly as stable as His-tRNA His containing G À1 in buffer at pH 7.5, and is slightly more stable at pH 4.5 (Fig. 6) mutation of the GUG anticodon drastically reduces Thg1 G À1 addition activity in vitro (Jackman and Phizicky 2006a). Moreover, suppression almost certainly occurs by insertion of histidine, since the Kex2 serine protease requires histidine at the catalytic H213 position, and since suppression requires overexpression of HTS1.…”

“…To study the function of the G À1 residue of tRNA His in the absence of Thg1 G À1 addition activity, we took advantage of the observation that the anticodon of tRNA His is necessary and sufficient for tRNA His guanylyltransferase activity by Thg1 (Jackman and Phizicky 2006a), and devised a tRNA His nonsense suppressor screen. Since Thg1 specifically recognizes the tRNA His GUG anticodon, and a tRNA His nonsense suppressor has a different anticodon (Fig.…”

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

“…Since tRNA identity is often associated with the anticodon, we tested the role of the GUG anticodon using tRNA His variants in which the GUG anticodon was replaced with the GAA sequence of tRNA Phe (Jackman and Phizicky 2006a). Both AcaHisRS and TbrHisRS are strongly dependent on the tRNA His anticodon, GUG (Fig.…”

Unexpected expansion of tRNA substrate recognition by the yeast m1G9methyltransferase Trm10

“…For enzymes that only act on a single tRNA species, recognition of the correct tRNA substrate is readily accomplished by distinct sequence elements, such as the tRNA His anticodon that is the critical element recognized by the tRNA His -specific guanylyltransferase, Thg1 (Jackman and Phizicky 2006). However, enzymes that modify multiple tRNA substrates face a more challenging task: How are specific substrates recognized from among a pool of highly similar tRNA species?…”