Shifted positioning of the anticodon nucleotide residues of amber suppressor tRNA species by Escherichia coli arginyl-tRNA synthetase

Cytidine in the anticodon second position (position 35) and G or U in posit ion 36 of tRNA(Arg) are required for aminoacylation by arginyl-tRNA synthet ase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting ambe r suppressor tRNA with a CUA anticodon (FTOR1 Delta 26) exhibits suppressio n activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804-1807 ]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1 Delta 26 involves C34 and U35, and that U35 can be re placed by G without affecting the activity. Thus, the positioning of the es sential nucleotides for the arginylation is shifted to the 5' side, by one residue, in the suppressor tRNA(Arg). We found that the shifted positioning does not depend on the tRNA sequence outside the anticodon. Furthermore, b y a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1 Del ta 26 more efficiently than the wild-type ArgRS. The isolated mutant has mu tations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site.