Molecular consequences of Ds insertion into and excision from the helix-loop-helix domain of the maize R gene

The R and B proteins of maize are required to activate the transcription of several genes in the anthocyanin biosynthetic pathway. To determine the st ructural requirements for R function in vivo, we are exploiting its sensiti ve mutant phenotype to identify transposon (Ds) insertions that disrupt cri tical domains. Here we report that the ability of the r-m1 allele to activa te transcription of at least three structural genes is reduced to only 2% o f wild-type activity because of a 396-bp Ds element in helix 2 of the basic helix-loop-helix (bHLH) motif. Residual activity likely results from the s ynthesis of a mutant protein that contains seven additional amino acids in helix 2. This protein is encoded by a transcript where most of the Ds seque nce has been spliced from pre-mRNA. Two phenotypic classes of stable deriva tive alleles, very pale and extremely pale, condition <1% of wild-type acti vity as a result of the presence of two- and three-amino-acid insertions, r espectively, at the site of Ds excision. Localization of these mutant prote ins to the nucleus indicates a requirement for an intact bHLH domain after nuclear import. The fact that deletion of the entire bHLH domain has only a minor effect on R protein activity while these small insertions virtually abolish activity suggests that deletion of the bHLH domain may bypass a req uirement for bHLH-mediated protein-protein interactions in the activation o f the structural genes in the anthocyanin biosynthetic pathway.