An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger.
The DNA binding domain of Adr1, the protein derived from alcohol dehydrogenase regulatory gene 1, is unusual for zinc finger proteins in that it consists of two classical Cys2His2 zinc fingers and an amino-terminal proximal accessory region termed PAR. PAR is unstructured in the free protein and becomes structured in the DNA-bound form. We investigated the role of PAR in DNA binding using molecular and biochemical approaches, and its importance for activation in vivo, using Adr1-dependent reporter genes. PAR was unimportant for DNA binding when a third finger was added to Adr1, and its importance was diminished but not eliminated by mutations in finger two that increased DNA binding affinity. The kinetic rate constants for three Adr1 proteins containing or lacking PAR were determined by surface plasmon resonance. PAR increased the on rate and decreased the off rate for specific DNA sites for Adr1 containing wild-type fingers one and two. Surprisingly, PAR had no significant effect on the kinetic rate constants when a third finger was present, or when single-stranded DNA was used as the substrate for DNA binding. A mutant form of Adr1-F1F2 in which finger 2 makes three base-specific contacts with DNA had a higher affinity for DNA than Adr1 containing three fingers, yet the mutant protein still depended on PAR for optimal binding affinity. The ability to activate transcription in vivo was correlated with a low dissociation rate, suggesting that stabilizing an activator at the promoter might be rate-limiting for transcription in vivo. PAR may have evolved to lend additional stability to DNA-Adr1 complexes encompassing short binding sites. In addition, PAR may have a role in transcription at a step after DNA binding since deletion of PAR from Adr1 with three fingers decreased activation in vivo but had no effect on DNA binding kinetics.
Young
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ET
Department of Biochemistry, Box 357350, University of Washington, Seattle, Washington 98195-7350, USA. ety@u.washington.edu
Kacherovsky
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N
Cheng
C
C
D000426:Alcohol Dehydrogenase / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005809:Genes, Regulator; D007700:Kinetics; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers
C074682:ADR1 protein, S cerevisiae; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D000426:Alcohol Dehydrogenase