Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.
Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that 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 importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, 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 phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.
Cheng
C
C
Department of Biochemistry, University of Washington, Seattle 98195.
Kacherovsky
N
N
Dombek
K M
KM
Camier
S
S
Thukral
S K
SK
Rhim
E
E
Young
E T
ET
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000478:Alkylation; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*
C074682:ADR1 protein, S cerevisiae; D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000426:Alcohol Dehydrogenase