IDENTIFICATION OF POTENTIAL TARGET GENES FOR ADR1P THROUGH CHARACTERIZATION OF ESSENTIAL NUCLEOTIDES IN UAS1

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae th at binds to and activates transcription from two sites in a perfect 22 -bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The impo rtance for DNA binding of each position within UAS1 was deduced from t wo types of assays. Both methods led to an identical consensus sequenc e containing only four essential base pairs: GG(A/G)G. The preferred s equence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for pho sphate contacts in the central region of UAS1. However, no base-specif ic contacts in this portion of UAS1 are important for DNA binding or f or ADR1-dependent transcription in vivo. When the central 6 bp were de leted, only a single monomer of Adr1p was able to bind in vitro and ac tivation in vivo was severely reduced. On the basis of these results a nd previous knowledge about the DNA binding site requirements, includi ng constraints on the spacing and orientation of sites that affect act ivation in vivo, a consensus binding site for Adr1p was derived. By us ing this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In ad dition, this consensus allowed the identification of new potential tar get genes for Adr1p.