DEVELOPMENT OF FLUORESCENTLY LABELLED MONONUCLEOSOMES TO STUDY TRANSCRIPTION MECHANISMS BY METHOD OF MICROSCOPY OF SINGLE COMPLEXES

Fluorescence microscopy of single molecules and complexes is becoming increasingly popular method to study nucleosomes and functionally important processes with their participation. In this work we report on the development of positioned mononucleosomes on the basis of fluorescently labeled DNA matrix, which are a new tool for investigation of structural rearrangements in chromatin during transcription by RNA polymerase (RNAP). Two fluorophores, Cy3 (donor) and Cy5 (acceptor), were introduced in a non-transcribed DNA chain. After DNA packing on histone core they are positioned in the middle part of a nucleosome in the neighboring supercoils at a distance of less than 60 Å and interact by the mechanism of Förster resonance energy transfer (FRET). Changes in the nucleosome structure were monitored by changes in FRET efficiency in the study of single complexes of nucleosomes with RNAP by fluorescence microscopy. It is shown that introduction of labels did not affect the ability of RNAP to transcribe nucleosomes. Open complex with RNAP as well as elongation complexes stalled at positions –39 and –5 before the nucleosome boundary were prepared and characterized. It is shown that more than 80% of histones maintain their structure, restoring the original packing of DNA on the histone core after transcription completion. A new experimental system offers new possibilities for the study of a structure of nucleosomes and modulating effects of various protein chaperones
and remodelers on them.

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