Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy
| Autor: | M. E. Valieva, K.S. Kudryashova, O. V. Chertkov, Vasily M. Studitsky, D. V. Nikitin, Alexey V. Feofanov, N. S. Gerasimova |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
030102 biochemistry & molecular biology Biology Linker DNA Molecular biology General Biochemistry Genetics and Molecular Biology Chromatin remodeling Chromatin 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Histone chemistry RNA polymerase Histone methylation biology.protein Biophysics Nucleosome Histone octamer General Agricultural and Biological Sciences General Environmental Science |
| Zdroj: | Moscow University Biological Sciences Bulletin. 70:189-193 |
| ISSN: | 1934-791X 0096-3925 |
| DOI: | 10.3103/s0096392515040069 |
| Popis: | 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 A, and, therefore, Forster 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. |
| Databáze: | OpenAIRE |
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