Dipeptide formation with misacylated tRNAPhes.
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.
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004151:Dipeptides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D012270:Ribosomes / Q000201:enzymology; D013329:Structure-Activity Relationship
D004151:Dipeptides; D012346:RNA, Transfer, Amino Acyl; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase