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Moscow University Biological Sciences Bulletin

, Volume 70, Issue 4, pp 189–193 | Cite as

Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy

  • K. S. Kudryashova
  • D. V. Nikitin
  • O. V. Chertkov
  • N. S. Gerasimova
  • M. E. Valieva
  • V. M. Studitsky
  • A. V. FeofanovEmail author
Methods

Abstract

Fluorescence microscopy of single molecules and complexes is an increasingly popular method for research on nucleosomes and functionally important processes involving these biological objects. Precisely positioned mononucleosomes have been developed in the present work using a fluorescently labeled DNA template; such nucleosomes are novel tools for the investigation of structural rearrangements of chromatin during transcription by RNA polymerase (RNAP). Two fluorophores, the donor Cy3 and the acceptor Cy5, were introduced into the nontranscribed DNA strand. DNA coiling around the histone octamer resulted in the positioning of both fluorophores on adjacent DNA coils in the middle part of the nucleosome. The distance between the fluorophores was less than 60 Å, and, therefore, Förster resonance energy transfer (FRET) could occur. Structural rearrangements in the nucleosomes were detected using the changes in FRET efficiency measured in fluorescence microscopic studies of individual complexes of nucleosomes with RNAP. Labeling had no effect on the ability of RNAP to transcribe DNA in nucleosomes. An open complex with RNAP and elongation complexes arrested in positions–39 and–5 relatively to the nucleosome border were obtained and characterized. More than 80% of the nucleosomes have been shown to retain their structure (that is, recover the initial positioning of DNA on the histone octamer) after the completion of transcription. The experimental system developed opens up new possibilities for research on nucleosome structure and its modulation by various protein chaperones and chromatin remodeling complexes.

Keywords

nucleosome RNA polymerase fluorescence microscopy epigenetics 

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Copyright information

© Allerton Press, Inc. 2015

Authors and Affiliations

  • K. S. Kudryashova
    • 1
    • 3
  • D. V. Nikitin
    • 3
  • O. V. Chertkov
    • 1
    • 3
  • N. S. Gerasimova
    • 3
  • M. E. Valieva
    • 3
  • V. M. Studitsky
    • 2
    • 3
  • A. V. Feofanov
    • 1
    • 3
    Email author
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesMoscowRussia
  2. 2.Cancer Epigenetics ProgramFox Chase Cancer CenterPhiladelphiaUnited States
  3. 3.Department of BiologyMoscow State UniversityMoscowRussia

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