Dipeptide formation with misacylated tRNAPhes

• Published in: The Journal of biological chemistry Vol. 258; no. 7; pp. 4492 - 4495
• Main Authors: Heckler, T G, Zama, Y, Naka, T, Hecht, S M
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.04.1983; American Society for Biochemistry and Molecular Biology
• Subjects: Acylation; Amino Acyl-tRNA Synthetases - metabolism; Dipeptides - biosynthesis; Escherichia coli - enzymology; Kinetics; Phenylalanine-tRNA Ligase - metabolism; Ribosomes - enzymology; RNA, Transfer, Amino Acyl - genetics; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)32650-4

Several misacylated P-site tRNAs have been prepared by the T4 RNA ligase-mediated coupling of Escherichia coli tRNAPhe missing the 3'-terminal cytidine and adenosine moieties and N-acetylaminoacyl-pCpA derivatives. The reaction proceeded in reasonable yield in each case and the tRNA products were purified conveniently by successive chromatographies on DEAE-cellulose and benzoylated DEAE-cellulose. The misacylated tRNAPhes were assayed for participation in the peptidyltransferase reaction, using E. coli ribosomes and poly(U) as message. When phenylalanyl-tRNAPhe was employed as the A-site tRNA, "chemically aminoacylated" N-acetyl-L-phenylalanyl-tRNAPhe produced dipeptide to virtually the same extent as authentic N-acetyl-L-phenylalanyl-tRNAPhe, which was prepared by enzymatic activation of tRNAPhe followed by chemical acetylation. Significant dipeptide formation was also obtained with N-acetyl-L-tyrosyl-tRNAPhe, but much less dipeptide was obtained when the D-isomers of these two N-acetylaminoacyl-tRNAs were employed in the same assay system. Interestingly, N-acetyl-beta-phenylalanyl-tRNAPhe formed dipeptide at least to the same extent as authentic N-acetyl-L-phenylalanyl-tRNAPhe.