AMINOACYL-TRANSFER-RNA RECOGNITION BY THE LEUCYL PHENYLALANYL-TRANSFER-RNA-PROTEIN TRANSFERASE/

We employ mutant and mischarged aminoacyl-tRNAs to characterize aminoa cyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transfera se (L/F-transferase). Wild type Met-tRNA(Metm) (CAU anticodon) and mis charged Met-tRNA(Val-1) (CAU anticodon) are substrates for the L/F-tra nsferase during the NH2-terminal aminoacylation of alpha-casein, where as Val-tRNA(Val-1) (UAC), Val-tRNA(Metm) (UAC), and Arg-tRNA(Metm) (CC G, A20) are not. Mutations in the anticodon and extra arm of tRNA(Leu- 1) do not measurably effect its ability to serve as a substrate for th e L/F-transferase, and the dissociation constants of the complexes bet ween L/F-transferase and either wild type Leu-tRNA(Leu-4) (UAA) or mut ant Leu-tRNA(Leu-4) (CUA) are each 0.4 +/- 0.2 mu M. The dissociation constants for the complexes between the L/F-transferase and uncharged tRNA, leucine methyl ester, and puromycin are all 10-1,000-fold greate r than that of the Leu-tRNA L/F-transferase complex. Dissociation of t he Leu-tRNA L/F-transferase complex is slow, relative to the rate calc ulated assuming that association is diffusion controlled, Finally, deo xyoligonucleotide aminoacyl-tRNA hybrids (dO . AA-tRNAs) are employed to characterize the determinants of the Leu-tRNA(Leu-4) acceptor stem recognized by the L/F-transferase. A dO . AA-tRNA completely lacking a cceptor stem base pairs remains a substrate for the L/F-transferase, w hereas a dO . AA-tRNA containing a a-base pair single-stranded region, at its 3' terminus, does not.