Use of Protein Synthesis In Vitro to Study Codon Recognition by Escherichia coli tRNALeu Isoaccepting Species

Abstract

In the past, studies of codon recognition by tRNA isoaccepting species have employed the techniques of ribosome binding (Nirenberg et al. 1967), protein synthesis in vitro directed by synthetic polyribonucleotides (Khorana et al. 1967), and direct nucleotide interactions between tRNAs containing complementary anticodons (Grosjean et al. 1976). The availability of completely sequenced, natural mRNAs has facilitated the study of codon recognition in vitro under conditions that more closely resemble the situation in vivo than were previously possible. One can now compare the relative efficiencies of different acylated tRNA isoaccepting species to transfer their amino acids into isolated peptides of known sequence that contain residues encoded by known codons. The efficiency of codon recognition is thus deduced from a functional test involving synthesis of a bona fide natural polypeptide chain directed by a natural mRNA.

The validity of this approach depends upon the presumptions that the amino acid isolated in a single peptide is in fact transferred only from the individual aminoacylated tRNA isoaccepting species added to the reaction and that no exchange of amino acid occurs between different isoaccepting species present in the extract. This constraint has been satisfied by two different approaches. The first is the use of extracts of Escherichia coli mutants that are temperature-sensitive in one of the aminoacyl-tRNA synthetase enzymes (Mitra et al. 1977). These extracts are dependent on added aminoacylated tRNA for protein synthesis to occur, and no exchange of the homologous amino acid should be possible. The second approach is the addition of a...