Studies on Polynucleotides: LXXXIX. A Study of Amino Acid Incorporation in a Reticuloctye Cell-Free Protein-Synthesizing System with Polyribonucleotides with Repeating Nucleotide Sequences used as Messengers

Abstract

Abstract A cell-free protein-synthesizing system from rabbit reticulocytes has been used to study amino acid incorporation with the use of polyribonucleotides of defined sequences as messengers. The following polyribonucleotide messengers were tested, and the amino acids incorporated in response to the corresponding messenger are shown in parentheses: poly r-U (Phe), poly r-A (Lys), poly r-C (Pro), poly r-UG (Val, Cys), poly r-AC (His, Thr), poly r-UC (Leu, Ser), poly r-AUC (His, Ile, Ser), poly r-GUA (Ser, Val), poly r-AUG (Asp, Met), poly r-CAA (Asn, Gln, Thr), and poly r-UUG (Val, Cys, Leu). No amino acid was incorporated in response to UAG and UGA codons. Reticulocyte ribosome preparations, after preincubation in the cell-free protein-synthesizing system to remove endogenous messenger RNA and washing with glycerol, still retained most of the enzymic activities required for protein synthesis. Such ribosome preparations were almost free of endogenous amino acids and transfer RNA. When tested in the presence of the above mentioned polyribonucleotide messengers but in the absence of lysate, these ribosome preparations gave amino acid incorporations similar to those described above. In addition, particularly in the presence of high Mg++ concentration, poly r-UG stimulated incorporation of methionine and tyrosine in addition to valine and cysteine and poly r-UUG stimulated incorporation of isoleucine and tyrosine in addition to valine, leucine, and cysteine. Competition experiments with unlabeled amino acids allowed the following ambiguous codon assignments: GUG for methionine, UGU for tyrosine, and GUU for isoleucine.