Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription

The role of the eucaryotic T-A-T-A box (Hogness box) homology sequence located approximately 30 base pairs upstream from the RNA initiation site has been further examined by oligodeoxynucleotide-directed site-specific mutagenesis. A method was employed which allows insertion of nucleotide-specific mutations into virtually any double-stranded, recombinant plasmid DNA. A synthetic mixed oligonucleotide, bearing defined multiple nucleotide substitutions at a single site, was used both as a specific mutagen during primed DNA repair synthesis in vitro, as well as a highly sensitive hybridization probe for the identification of the mutated cloned DNA. Using this methodology, an A leads to G transition mutation was introduced into the second position of the ovalbumin gene T-[A]-T-A box, and the effect of modifying this highly conserved nucleotide on the expression of the mutant DNA was analyzed in a cell-free transcription system. Comparison of two allelic ovalbumin genes, slightly divergent upstream from the TATA box, resulted in identical in vitro transcription efficiencies. While correct initiation of ovalbumin RNA transcripts was not affected, the efficiency of gene expression using the mutant template, compared to either the corresponding wild-type sequence or the allelic gene, was markedly reduced. These results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.