PubMed · 13444109
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J A BUTLER, P F DAVISON. 1957. Deoxyribonucleoprotein, a genetic material.. https://doi.org/10.1002/9780470122631.ch4
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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.
Mammalian RNA polymerase II transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase II. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase II, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase II.
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