Yokota H, Nickerson D A, Trask B J, van den Engh G, Hirst M, Sadowski I, Aebersold R
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA.
Anal Biochem. 1998 Nov 15;264(2):158-64. doi: 10.1006/abio.1998.2851.
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.
我们开发了一种基于原子力显微镜（AFM）的方法，用于在纳米分辨率下绘制单个长DNA分子（> 5 kb）上的蛋白质结合位点。在弯曲的DNA分子顶端能清晰检测到蛋白质。随机盘绕的DNA分子或蛋白质：DNA复合物通过原子级平整表面上由电机控制的移动弯月面进行伸展。通过AFM检测固定的分子。拉直的DNA在结合蛋白质的位点处呈现出尖锐的弯曲，两个DNA片段从蛋白质结合位点线性延伸。使用酵母转录因子GAL4，我们证明了在拉直的DNA模板上观察到的结合位点位置与预测的结合位点具有良好的一致性。该技术有望在阐明DNA和蛋白质的相互作用方面，特别是在单分子水平上测量未标记调节蛋白对启动子的占据情况方面，具有重要意义。
