The synthesis and cloning of two tyrosine suppressor tRNA genes with altered promoter sequences.

The total synthesis of the suppressor tRNA gene (ssu1) containing a 51-base pair long "natural" promoter has previously been reported. In this paper, we describe the synthesis and characterization of two genes (ssu3 and ssu4) in which the promoter sequence present in the parent ssu1 gene has been altered. The modifications, introduced as a part of the study of structure-function relationships, were placed in the two known prominent regions of homology. The change introduced in ssu3 was a G:C to A:T transition at -10 nucleotide, while the two changes incorporated in ssu4 involved a G:C to A:T transition at -36 nucleotide and a G:C to T:A transversion at -38 nucleotide. The promoters containing the modifications were constructed by T4-polynucleotide ligase joining of the appropriate chemically synthesized oligonucleotides. These promoters then replaced the promoter region of the cloned and previously synthesized ssu2 gene by utilization of the HindIII site present at the junction of the promoter and the structural gene. The genes containing the modified promoters have been cloned into the plasmid vector pBR322 by insertion into the unique Eco RI site. The DNA sequencing of the cloned genes now described as well as that of the previously cloned and chemically synthesized genes (ssu1 and ssu2) provide direct confirmation of the accuracy of the synthetic work (chemical and enzymatic) at all stages.