Et. Young et al., An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger, BIOCHEM, 39(3), 2000, pp. 567-574
The DNA binding domain of Adr1, the protein derived from alcohol dehydrogen
ase regulatory gene 1, is unusual for zinc finger proteins in that it consi
sts of two classical Cys2His2 zinc fingers and an amino-terminal proximal a
ccessory region termed PAR. PAR is unstructured in the free protein and bec
omes structured in the DNA-bound form. We investigated the role of PAR in D
NA binding using molecular and biochemical approaches, and its importance f
or-activation in vivo, using Adr1-dependent reporter genes. PAR was unimpor
tant for DNA binding when a third finger was added to Adr1, and its importa
nce was diminished but not eliminated by mutations in finger two that incre
ased DNA binding affinity. The kinetic rate constants for three Adr1 protei
ns 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 n
o 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 bind
ing. 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 evo
lved 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 decrea
sed activation in vivo but had no effect on DNA binding kinetics.