N,iε-Acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acids

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Abstract
Unfractionated Escherichia coli tRNA was aminoacylated with lysine and preferentially acetylated at the .epsilon.-amino N of lysine by reaction with N-acetoxysuccinimide. After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were N.epsilon.-acetyl-Lys-tRNA. Post-ribosomal supernatant enzymes would not deacylate N.epsilon.-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which was exposed to the acetylation reaction conditions. Poly(rA) stimulated the binding of N.epsilon.-acetyl-Lys-tRNA to E. coli ribosomes. At the ribosome and tRNA concentrations used, N.epsilon.-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound 1/2 as well as Lys-tRNA. Both Lys-tRNA and N.epsilon.-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 30 mM Mg2+. When Lys-tRNA or N.epsilon.-acetyl-Lys-tRNA preparations from E. coli were added to rabbit reticulocyte cell-free protein synthesizing incubations, the incorporation of either amino acid into protein was complete within 5 min. The final incorporation level of the analogue was 82% that of the unmodified lysine. After protein synthesized in the presence of N.epsilon.-acetyl-[14C]Lys-tRNA was digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as N.epsilon.-acetyllysine. Gel filtration of the post-ribosomal supernatant revealed that most of the N.epsilon.-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin.

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