Undiscriminating codon reading with adenosine in the wobble position.

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.