Unfractionated Escherichia coli tRNA has been aminoacylated with lysine and preferentially acetylated at the e-amino nitrogen of lysine by reaction with Nacetoxysuccinimide. After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were Ac-acetyl-Lys-tRNA. Post-ribosomal supernatant enzymes would not deacylate 7Ve-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which had been exposed to the acetylation reaction conditions. Poly(rA) stimulated the binding of NE-acetyl-Lys-tRNA to E. coli ribosomes. At the ribosome and tRNA concentrations used, M-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound one-half as well as Lys-tRNA. Both Lys-tRNA and NE-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 50 mM Mg2+. When Lys-tRNAE, coil or NE-acetyl-Lys-tRN AE. 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 NE-acetyl- [14C] Lys-tRN A had been digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as (NE-acetyllysine. Gel filtration of the post-ribosomal supernatant revealed that most of the NE-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin.
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