Genetically and chemically derived missense suppressor transfer RNA's with altered enzymic aminoacylation rates

Abstract

A transfer RNA species with altered specificity in an in vitro amino acid-incorporating system has been obtained by partial deamination of tRNA GGA Gly with nitrous acid. This altered tRNA inserts [ 14C]glycine into polypeptides in response to the alternating polyribonucleotide, poly (A-G), an activity previously shown to be characteristic of tRNA from Escherichia coli strains carrying a suppressor of mutation A36 in the tryptophan synthetase A protein. Both the genetically ( su 36 +) and chemically derived suppressor tRNA's read the arginine codon, AGA, as a glycine codon. Improvements in the method of preparation of the chemically derived “suppressor” have established that the activity arises from a change in a tRNA GGA Gly. An anticodon change (UCC→UCU) is probably involved, but direct evidence of this is still lacking. Chromatographic comparisons of the genetically and chemically derived suppressors have shown them to be different molecular species, although both chromatograph in the tRNA Gly region in two systems. Previous indications that the su 36 + suppressor reacts slowly in the aminoacylation reaction catalyzed by the glycine activating enzyme have been confirmed in vitro. The actual rates of enzymic charging of the two suppressors have been measured by making use of periodate oxidation to inactivate that fraction not aminoacylated after a given time in loading reactions catalyzed by various levels of purified glycyl tRNA synthetase. It requires approximately 4 × 10 5 times more enzyme to load these suppressors to completion than is required to aminoacylate the same quantity of normal tRNA Gly in 30 minutes at 37 °C. When corrected for concentration effects, it appears that both the su 36 + and chemically derived suppressor tRNA's react approximately 2 × 10 3 times slower than does normal tRNA Gly in the enzymic aminoacylation reaction (at saturating levels of tRNA).