SOME DNA TARGETS OF THE YEAST CYP1 TRANSCRIPTIONAL ACTIVATOR ARE FUNCTIONALLY ASYMMETRIC - EVIDENCE OF 2 HALF-SITES WITH DIFFERENT AFFINITIES

CYP1 protein is a yeast transcriptional regulator which contains a zin c cluster in its DNA-binding domain. It was recently shown by selectin g random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Nota bly, this ideal sequence is however not found in wild-type CYP1 target sites. In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS 1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purifi ed CYP1-(1-200)-peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA , both CGGs are essential for binding. (b) If the sequence element con tains only the right CGG and the TA, both are sufficient but indispens able for the binding of CYP1. (c) When two CGCs and the TA are present , the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1-(1-200)-peptide is usually a monomer in solution but b inds DNA as a dimer. Finally, we found evidence for the presence of tw o half-sites with different measured affinities in the asymmetric sequ ences of some CYP1 targets.