Mapping a protein-binding site on straightened DNA by atomic force microscopy.
We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.
Yokota
H
H
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA.
Nickerson
D A
DA
Trask
B J
BJ
van den Engh
G
G
Hirst
M
M
Sadowski
I
I
Aebersold
R
R
D000538:Aluminum Silicates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D013500:Surface Tension; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000538:Aluminum Silicates; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; C011934:mica