Studies on polynucleotides: LXVII. Initiation of protein synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide sequences as messengers

Abstract

All of the 64 ribotrinucleotides were tested for the binding of fmet-tRNA F to ribosomes. The trinucleotides AUG, GUG, UUG, ACG, CUG, GCG, UGG, UCG and AGG stimulated the binding, the first three giving the maximum effect. The non-formylatable species (met-tRNA M) was recognized by the triplets AUG and GUG. Polypeptide synthesis in Escherichia coli cell-free system at 0.004 m-Mg 2+ concentration proceeded only when messengers contained initiator codons; it also required the presence of fmet-tRNA F and additional stimulation was obtained by the addition of a protein fraction isolated from ribosomal washings. Poly r-UG containing repeating dinucleotide sequence directed the synthesis of fmet-(Cys-Val-) n , and the N-terminal sequence was established as fmet-Cys-Val-Poly r-AUG directed polymethionine synthesis required fmet-tRNA F in addition to met-tRNA M needed for the incorporation of internal methionine. At low Mg 2+ ion concentration poly r-GUA specifically stimulated the incorporation of valine, dependent on fmet-tRNA F. Although the triplet UUG stimulated the binding of fmet-tRNA F to ribosomes, poly r-UUG failed to stimulate fmet incorporation and no fmet-tRNA F-dependent polypeptide synthesis at 0.004 m-Mg 2+ concentration was observed. From these results the triplets AUG, GUG and GUA are concluded to be codons for the initiation of polypeptide chains by fmet-tRNA F.