Bhaumik Sukesh R
Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL-62901, USA.
Methods. 2006 Dec;40(4):353-9. doi: 10.1016/j.ymeth.2006.06.025.
Gene expression in eukaryotes is largely controlled at the level of transcriptional initiation by gene-specific activators. Transcriptional activators stimulate the assembly of general transcription factors on the promoter to form a preinitiation complex (PIC). Such a stimulated assembly of PIC is believed to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the "target". Based primarily upon in vitro protein-protein interaction experiments, a variety of factors have been proposed to be the direct targets of activators. However, whether any of these are bona fide in vivo targets required for stimulation of PIC assembly and hence transcriptional activation remains mostly unknown, primarily because of lack of appropriate experimental methods. Using a confocal microscopy-based FRET (fluorescence resonance energy transfer) assay, we have recently identified the target of a prototypic activator, Gal4p, in living yeast cells. Here, we describe the FRET assay in general for analysis of the targets of transcriptional activators in living yeast cells. Such an assay can also be used as a general method to monitor protein-protein interactions in vivo.
真核生物中的基因表达在很大程度上是由基因特异性激活因子在转录起始水平上控制的。转录激活因子刺激通用转录因子在启动子上组装，形成预起始复合物（PIC）。据信，PIC的这种受刺激组装是由激活因子与转录机制的一个或多个组分（称为“靶点”）之间的直接相互作用导致的。主要基于体外蛋白质-蛋白质相互作用实验，已经提出了多种因子作为激活因子的直接靶点。然而，这些因子中是否有任何一个是刺激PIC组装并因此激活转录所必需的真正体内靶点，仍然大多未知，主要是因为缺乏合适的实验方法。利用基于共聚焦显微镜的荧光共振能量转移（FRET）分析，我们最近在活酵母细胞中鉴定出了原型激活因子Gal4p的靶点。在这里，我们总体描述用于分析活酵母细胞中转录激活因子靶点的FRET分析。这种分析也可以用作监测体内蛋白质-蛋白质相互作用的通用方法。
