Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins

Abstract

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.