The Enzymic Synthesis of Amino Acyl Derivatives of Ribonucleic Acid

The previous papers (l-3) and reports from numerous laboratories (4-9) have demonstrated the enzymic synthesis of amino acyl ribonucleic acid derivatives. Studies by Zachau et al. (lo), Hecht et al. (ll), and ourselves (3) have established that each amino acid is linked to a specific polynucleotide chain through an ester linkage with either the 2’or 3’-hydrosyl group of the terminal nucleotide residue. In each instance where it has been investigated, the terminal nucleotide has been identified as adenylic acid (3, 10-12). In the amino acid-acceptor ribonucleic acid from mammalian liver it was reported that the adenylic acid unit is linked to a dinucleotide sequence of two cytidylic acid residues (11). In an attempt to define the nucleotide sequence required for amino acid acceptor activity, we have investigated methods for partially degrading the RNA chains and the requirements for reconverting such degraded preparations to their native state. Removal of one or a few nucleotides from the 3’-hydroxyl end of the polynucleotide chains with purified venom phosphodiesterase destroys the amino acid acceptor activity (3). Such a partially degraded RNA preparation, but not the native material, is an excellent primer for the incorporation of (Flabeled adenosine phosphate and Ci4-labeled cytidine phosphate from their respective triphosphates by an enzyme obtained from Escherichia coli. Depending upon the extent of degradation of the RNA, the purified enzyme forms part or all of the terminal trinucleotide sequence . . . pCpCpAl resulting in the restoration of amino acid acceptor activity. The incorporation of each nucleotide unit is accompanied by the liberation of one equivalent of inorganic pyrophosphate. As might be predicted from this finding, the enzyme catalyzes a net pyrophosphorolysis of the intact amino acid-acceptor RNA yielding adenosine triphosphate and cytidine triphosphate from the terminal trinucleotide. The studies reported here are in agreement with the findings of Hecht et al. (14). These workers reported that after inactivation of mammalian amino acid-acceptor RNA by preincubation with a “pH 5 fraction” from liver or ascites cell extracts,