Evans Sara K, Aiello David P, Green Michael R
Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
Biochem Soc Symp. 2006(73):217-24.
The first step in transcriptional activation of protein-coding genes involves the assembly on the promoter of a large PIC (pre-initiation complex) comprising RNA polymerase II and a suite of general transcription factors. Transcription is greatly enhanced by the action of promoter-specific activator proteins (activators) that function, at least in part, by increasing PIC formation. Activator-mediated stimulation of PIC assembly is thought to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the 'target'. The unambiguous identification of direct, physiologically relevant in vivo targets of activators has been a considerable challenge in the transcription field. The major obstacle has been the lack appropriate experimental methods to measure direct interactions with activators in vivo. The development of spectral variants of green fluorescent protein has made it possible to perform FRET (fluorescence resonance energy transfer) analysis in living cells, thereby allowing the detection of direct protein-protein interactions in vivo. Here we discuss how FRET can be used to identify activator targets and to dissect in vivo mechanisms of transcriptional activation.
蛋白质编码基因转录激活的第一步涉及在启动子上组装一个大型的预起始复合物（PIC），该复合物由RNA聚合酶II和一套通用转录因子组成。启动子特异性激活蛋白（激活剂）的作用可极大地增强转录，这些激活剂至少部分通过增加PIC的形成来发挥作用。激活剂介导的PIC组装刺激被认为是由于激活剂与转录机器的一个或多个组分（称为“靶点”）之间的直接相互作用所致。在转录领域，明确鉴定激活剂在体内直接的、生理相关的靶点一直是一项重大挑战。主要障碍在于缺乏合适的实验方法来测量体内与激活剂的直接相互作用。绿色荧光蛋白光谱变体的发展使得在活细胞中进行荧光共振能量转移（FRET）分析成为可能，从而能够检测体内直接的蛋白质-蛋白质相互作用。在此，我们讨论如何利用FRET来鉴定激活剂靶点并剖析转录激活的体内机制。
