Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET

Kseniya S. Kudryashova, Oleg V. Chertkov, Dmitry V. Nikitin, Nikolai A. Pestov, Olga I. Kulaeva, Anastasija V. Efremenko, Alexander S. Solonin, Mikhail P. Kirpichnikov, Vasily M. Studitsky, Alexey V. Feofanov

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

4 Scopus citations

Abstract

Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

Original languageEnglish
Title of host publicationChromatin Protocols
Subtitle of host publicationThird Edition
PublisherSpringer New York
Pages395-412
Number of pages18
Volume1288
ISBN (Electronic)9781493924745
ISBN (Print)9781493924738
DOIs
StatePublished - Mar 31 2015

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Keywords

  • DNA-Directed RNA Polymerases/metabolism
  • Fluorescence Resonance Energy Transfer/methods
  • In Vitro Techniques
  • Nucleosomes/genetics
  • Transcription, Genetic

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