Analysis of Structure of Elongation Complexes in Polyacrylamide Gel with Förster Resonance Energy Transfer Technique

To study the structural features of elongation complexes (EC) formed during nucleosome transcription by RNA polymerase II (Pol II), it was proposed to use an approach based on the measurement of the efficiency of Förster resonance energy transfer (FRET) in polyacrylamide gel after separation of transcription intermediates by electrophoresis. Fluorescently labeled nucleosomes were obtained by introducing a pair of fluorophores (donor and acceptor) into adjacent gyres of nucleosomal DNA. Stalled ECs were formed by transcription of nucleosomes in the presence of a limited set of nucleoside triphosphates. Comparative analysis of FRET efficiency for free nucleosomes and ECs with Pol II obtained on their basis revealed changes in the structure of nucleosomes in ECs. It was found that ECs with the same RNA length can exist in different conformational states.