Studies on polynucleotides: LXXIV. Direct translation in vitro of single-stranded DNA-like polymers with repeating nucleotide sequences in the presence of neomycin B

Abstract

The messenger activity of single-stranded deoxyribopolynucleotides containing repeating nucleotide sequences, poly d-A, poly d-T, poly d-CA and poly d-TG, has been examined in the presence of the antibiotic neomycin B in the cell-free protein-synthesizing system from Escherichia coli B. Except for poly d-A, which failed to stimulate the incorporation of any amino acid, excellent incorporations were observed with the other deoxyribopolynucleotides. The amino acids incorporated were the same as expected for the corresponding ribopolynueleotides but in the absence of neomycin B. However, cases of occasional specific misreading, in general at very low efficiency relative to normal incorporations, were observed. Thus, poly d-CA stimulated the incorporation of only two amino acids, threonine and histidine. The product of the reaction was characterized as a copolypeptide containing threonine and histidine in alternating sequence. Poly d-TG stimulated the incorporation of the expected amino acids, cysteine and valine, that of alanine and arginine to a lesser extent and those of glutamic acid and glycine to a slight extent. Poly d-T stimulated mostly the incorporation of phenylalanine but slightly that of leucine. A number of related antibiotics, excepting gentamicin, failed to stimulate phenylalanine incorporation with poly d-T. Except for poly d-A, the deoxyribopolynucleotides stimulated the binding of the appropriate aminoacyl-tRNA's to ribosomes, and neomycin B was not required for this step. The initiator tRNA, formylmethionyl-tRNA, was also bound to ribosomes by poly d-TG: polypeptide synthesis directed by this deoxypolynucleotide in the presence of neomycin B at low magnesium ion concentration was dependent on the presence of the initiator-tRNA.