Journal
RNA
Volume 12, Issue 6, Pages 1007-1014Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.54706
Keywords
tRNA(His); tRNA(His) guanylyltransferase; anticodon; tRNA synthetase; yeast
Categories
Funding
- NIGMS NIH HHS [R01 GM052347, GM52347] Funding Source: Medline
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM052347] Funding Source: NIH RePORTER
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All eukaryotic tRNA(His) molecules are unique among tRNA species because they require addition of a guanine nucleotide at the -1 position by tRNA(His) guanylyltransferase, encoded in yeast by THG1. This G(-1) 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 tRNA(His) 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 tRNA(Phe). 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.
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