Total synthesis of a tyrosine suppressor tRNA gene. XVIII. Biological activity and transcription, in vitro, of the cloned gene.

Abstract

The chemically synthesized gene for Escherichia coli tyrosine suppressor tRNA has been joined to both plasmid (ColE1 ampr) and bacteriophage (Charon 3A) vector chromosomes after the latter had been digested with the restriction endonuclease EcoRI. Suppression of both bacterial (trpA, his, lacZ) and bacteriophage lambda amber mutations (Aam32, Bam1) has been demonstrated after transformation of E. coli with the recombinant DNA molecules carrying the synthetic suppressor tRNA gene. The cloned synthetic gene has been reisolated from the vector chromosomes after digestion of the latter with EcoRI restriction endonuclease and characterized in regard to its size and its ability to serve as a source of suppressor activity in further transformation experiments. This synthetic gene has also been shown to suppress bacterial amber mutations after it had been incorporated into the E. coli chromosome as part of a lambda prophage. Transcription, in vitro, of the cloned synthetic suppressor gene gave a product which, on treatment with a crude E. coli extract, afforded the tyrosine suppressor tRNA precursor. The latter was characterized by two-dimensional fingerprinting after digestion with T1-RNase. Exposure of the in vitro transcript to RNase P Selectively released the 41-nucleotide-long fragment characteristic of the 5'-end of the tRNA precursor. Thus, the nucleotide sequence of the cloned gene is accurate and its expression is controlled by its promoter.