Ribosomal synthesis of polyleucine on polyuridylic acid as a template

The ability of the ribosomal machinery to miscode was discovered more than 15 years ago. Incorporation of phenylalanine into peptide synthesized in a cell-free translation system with poly(U) was shown to be accompanied by incorporation of small amounts of leucine and isoleucine, amino acids close to phenylalanine in their code specificity [l-4]. It was also observed that in the absence of phenylalanine poly(U)programmed Escherichia coli ribosomes were capable of incorporating leucine [5,6] or isoleucine [7] into acid-insoluble product, presumably polyleucine or polyisoleucine, respectively. We have demonstrated ribosomal poly(U)-directed polyleucine incorporation in the E. coli cell-free system containing pure elongation factors (EF-Tu and EF-G) and leucyl-tRNA as the only substrate [8]. The product was identified as polyleucine using pepsin digestion and subsequent analysis of the oligopeptides produced [8]. Earlier it was shown that poly(U)-directed synthesis of polyphenylahmine can be performed by E. coli ribosomes in the absence of one or both elongation factors (EF-TU or/and EF-G) [9]. Here, in order to check possible contributions of the elongation factors to substrate selectivity, we have used one-factorpromoted translation systems, as well as factor-free translation systems for the poly(U)directed synthesis of polyleucine from leucyl-tRNA. We have demonstrated that poly(U)-programmed ribosomes synthesize polyphenylalanine -lo-30-times faster than polyleucine and have found that the presence of elongation factors does not contribute drastically to the selectivity of the ribosomal machinery at high [Mg’+] (210 mM);polyleucine synthesis isaccelerated by EF-Tu and/or EF-G to roughly the same extent