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 structural requirements for R function in vivo, we are exploiting its sensitive mutant phenotype to identify transposon (Ds) insertions that disrupt critical domains. Here we report that the ability of the r-m1 allele to activate transcription of at least three structural genes is reduced to only 2% of 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 synthesis 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 sequence has been spliced from pre-mRNA. Two phenotypic classes of stable derivative alleles, very pale and extremely pale, condition <1% of wild-type activity as a result of the presence of two- and three-amino-acid insertions, respectively, at the site of Ds excision. Localization of these mutant proteins 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 requirement for bHLH-mediated protein-protein interactions in the activation of the structural genes in the anthocyanin biosynthetic pathway.
Liu
Y
Y
Department of Botany, University of Georgia, Athens, Georgia 30602, USA.
Wang
L
L
Kermicle
J L
JL
Wessler
S R
SR
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements; D017343:Genes, Plant; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger; D018749:RNA, Plant; D013347:Subcellular Fractions; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D009687:Nuclear Proteins; D010940:Plant Proteins; C085200:R protein, Zea mays; D012333:RNA, Messenger; D018749:RNA, Plant