Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR.
The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.
Schaufler
Lawrence E
LE
Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA.
Klevit
Rachel E
RE
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
C074682:ADR1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA