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An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of site-specific mutations by using a mixture of oligodeoxyribonucleotides. With this method, as high as 40% of the tested clones turned out to be desired mutants. Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene. In vitro transcription experiments showed that single-base transversions at the TATA box (A----T at position -29, T----A or G at -28, A----T at -27, and A----T at -26) resulted in decreased promoter activities, whereas A----G transitions at positions -24 and -23 had no effect. The initiation site of transcription was normal in three down-promoter mutants at positions -28 and -26, but the A----T transversions at positions -29 and -27 induced an additional transcription start from position +4. Using 10 single-point mutants obtained as above or by nitrous acid-induced mutagenesis, we have prepared a pair of heteroduplex DNAs consisting of a mutant strand and the wild-type one. The molecules heterozygous at positions -30, -21, and -20 showed reduced transcription activities when the noncoding strand bears the mutation, whereas that at position -26 gave a low activity when the coding strand carries the mutation. Both types of heteroduplex at positions -29, -28, -27, and -17 exhibited decreased activities. These results suggest a transcription machinery contact to a major groove of the DNA helix at the TATA box and region of position -20.

Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants.