Aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase.
We employ mutant and mischarged aminoacyl-tRNAs to characterize aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase (L/Ftransferase). Wild type Met-tRNAMetm (CAU anticodon) and mischarged Met-tRNAVal-1 (CAU anticodon) are substrates for the L/F-transferase during the NH2-terminal aminoacylation of alpha-casein, whereas Val-tRNAVal-1 (UAC), Val-tRNAMetm (UAC), and Arg-tRNAMetm (CCG, A20) are not. Mutations in the anticodon and extra arm of tRNALeu-1 do not measurably effect its ability to serve as a substrate for the L/F-transferase, and the dissociation constants of the complexes between L/F-transferase and either wild type Leu-tRNALeu-4 (UAA) or mutant Leu-tRNALeu-4 (CUA) are each 0.4 +/- 0.2 microM. 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 greater than that of the Leu-tRNA.L/F-transferase complex. Dissociation of the Leu-tRNA.L/F-transferase complex is slow, relative to the rate calculated assuming that association is diffusion controlled. Finally, deoxyoligonucleotide.aminoacyl-tRNA hybrids (dO.AA-tRNAs) are employed to characterize the determinants of the Leu-tRNALeu-4 acceptor stem recognized by the L/F-transferase. A dO.AA-tRNA completely lacking acceptor stem base pairs remains a substrate for the L/F-transferase, whereas a dO.AA-tRNA containing a 2-base pair single-stranded region, at its 3' terminus, does not.
Abramochkin
G
G
Departments of Biochemistry and Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Shrader
T E
TE
D000217:Acyltransferases / Q000378:metabolism*; D019881:Aminoacyltransferases; D000926:Anticodon; D001483:Base Sequence; D001667:Binding, Competitive; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D017354:Point Mutation; D011691:Puromycin / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D000926:Anticodon; D012346:RNA, Transfer, Amino Acyl; D011691:Puromycin; D000217:Acyltransferases; D019881:Aminoacyltransferases; C021511:leucyltransferase