An Accessory DNA Binding Motif in the Zinc Finger Protein Adr1 Assists Stable Binding to DNA and Can Be Replaced by a Third Finger
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- 作者：Elton T. Young ; Nataly Kacherovsky ; and Cheng Cheng
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：January 25, 2000
- 年：2000
- 卷：39
- 期：3
- 页码：567 - 574
- 全文大小：105K
- 年卷期：v.39,no.3(January 25, 2000)
- ISSN：1520-4995
文摘
The DNA binding domain of Adr1, the protein derived from alcohol dehydrogenase regulatorygene 1, is unusual for zinc finger proteins in that it consists of two classical Cys2His2 zinc fingers andan amino-terminal proximal accessory region termed PAR. PAR is unstructured in the free protein andbecomes structured in the DNA-bound form. We investigated the role of PAR in DNA binding usingmolecular and biochemical approaches, and its importance for activation in vivo, using Adr1-dependentreporter genes. PAR was unimportant for DNA binding when a third finger was added to Adr1, and itsimportance was diminished but not eliminated by mutations in finger two that increased DNA bindingaffinity. The kinetic rate constants for three Adr1 proteins containing or lacking PAR were determined bysurface plasmon resonance. PAR increased the on rate and decreased the off rate for specific DNA sitesfor Adr1 containing wild-type fingers one and two. Surprisingly, PAR had no significant effect on thekinetic rate constants when a third finger was present, or when single-stranded DNA was used as thesubstrate for DNA binding. A mutant form of Adr1-F1F2 in which finger 2 makes three base-specificcontacts with DNA had a higher affinity for DNA than Adr1 containing three fingers, yet the mutantprotein still depended on PAR for optimal binding affinity. The ability to activate transcription in vivowas correlated with a low dissociation rate, suggesting that stabilizing an activator at the promoter mightbe 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 ata step after DNA binding since deletion of PAR from Adr1 with three fingers decreased activation invivo but had no effect on DNA binding kinetics.