Ramirez-Carrozzi V, Kerppola T
Department of Biological Chemistry, Howard Hughes Medical Institute, Ann Arbor, Michigan 48109-0650, USA.
Methods. 2001 Sep;25(1):31-43. doi: 10.1006/meth.2001.1213.
A gel-based fluorescence resonance energy transfer (gelFRET) assay was developed for analysis of the architecture of nucleoprotein complexes. gelFRET is based on fluorescence analysis of nucleoprotein complexes separated by polyacrylamide gel electrophoresis. These complexes are separated from free components and nonspecific complexes, enabling fluorescence analysis of complexes containing all components in stoichiometric proportions. gelFRET can be used to investigate the structural organization of nucleoprotein complexes through comparison of the relative efficiencies of energy transfer from donor fluorophores linked to different positions on DNA to an acceptor fluorophore linked to a unique position on the binding protein. We have applied gelFRET to analysis of the orientation of binding by heterodimeric transcription factors. By using Fos-Jun heterodimers as a model system we have identified the structural determinants that control the orientation of heterodimer binding. gelFRET can be applied to studies of a variety of biological processes that influence the proximity of two sites within a complex, such as the assembly of transcription regulatory complexes.
开发了一种基于凝胶的荧光共振能量转移（gelFRET）分析方法，用于分析核蛋白复合物的结构。gelFRET基于对通过聚丙烯酰胺凝胶电泳分离的核蛋白复合物的荧光分析。这些复合物与游离成分和非特异性复合物分离，从而能够对包含化学计量比例所有成分的复合物进行荧光分析。通过比较连接到DNA不同位置的供体荧光团到连接到结合蛋白上独特位置的受体荧光团的能量转移相对效率，gelFRET可用于研究核蛋白复合物的结构组织。我们已将gelFRET应用于异二聚体转录因子结合方向的分析。通过使用Fos-Jun异二聚体作为模型系统，我们确定了控制异二聚体结合方向的结构决定因素。gelFRET可应用于研究影响复合物内两个位点接近程度的各种生物过程，例如转录调节复合物的组装。
