Abstract
2' and 3'-O-(N-acetyl-L-phenylalanyl) adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by highperformance liquid chromatography (HPLC). From the two isomers of [3H] Phe-tRNA in equilibrium, Ac-[3H] Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H] Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H] Phe-tRNA was found to be 0.20:0.80. Further, [3H] Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[β, γ-imido] triphosphate (GMP-P (NH) P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H] Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H] Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu•GTP and 0.04:0.96 in the complex with EF-Tu•GMP-P (NH) P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.
