Fluorescence resonance energy transfer as a method for dissecting in vivo mechanisms of transcriptional activation.
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.
Evans
Sara K
SK
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.
Aiello
David P
DP
Green
Michael R
MR
D004268:DNA-Binding Proteins; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D012319:RNA Polymerase II