TY - GEN
T1 - Single-particle FRET microscopy of immobilized nucleosomes
T2 - 3rd International Multidisciplinary Microscopy Congress, InterM2015
AU - Feofanov, Alexey V.
AU - Chertkov, Oleg V.
AU - Kudryashova, Kseniya S.
AU - Ivanov, Yaroslav O.
AU - Studitsky, Vasily M.
AU - Kirpichnikov, Mikhail P.
N1 - Publisher Copyright:
© Springer International Publishing AG 2017.
PY - 2017
Y1 - 2017
N2 - Mononucleosomes formed by histone octamer and short DNA is an advanced model system for investigation of RNA polymerase (RNAP) transcription and its modulation with various transcription factors. Recent achievements of fluorescent microscopy allow one to complement these studies with single-particle Forster resonance energy transfer (spFRET) analysis. FRET efficiency between Cy3 and Cy5 dyes introduced in the neighboring coils of nucleosomal DNA is a sensor of structural changes caused by DNA unwrapping or looping, or restoration of DNA-histone interactions. Here we report on experimental setup and protocols for spFRET microscopy of immobilized nucleosomes. Using confocal laser scanning and total internal reflection fluorescence microscopy we demonstrated preservation of the nucleosome structure during immobilization and long-term (>100 s, ca. 140 ms/frame) spFRET kinetic measurements. The effect of ionic strength on nucleosome structure was studied. Applications of spFRET microscopy of immobilized nucleosomes include analysis of their structural dynamics (DNA “breathing”), kinetics of formation/dissociation of DNA-protein complexes, formation and structure of stalled elongation complexes with RNAP, as well as conformational transitions in nucleosome structure in the course of transcription.
AB - Mononucleosomes formed by histone octamer and short DNA is an advanced model system for investigation of RNA polymerase (RNAP) transcription and its modulation with various transcription factors. Recent achievements of fluorescent microscopy allow one to complement these studies with single-particle Forster resonance energy transfer (spFRET) analysis. FRET efficiency between Cy3 and Cy5 dyes introduced in the neighboring coils of nucleosomal DNA is a sensor of structural changes caused by DNA unwrapping or looping, or restoration of DNA-histone interactions. Here we report on experimental setup and protocols for spFRET microscopy of immobilized nucleosomes. Using confocal laser scanning and total internal reflection fluorescence microscopy we demonstrated preservation of the nucleosome structure during immobilization and long-term (>100 s, ca. 140 ms/frame) spFRET kinetic measurements. The effect of ionic strength on nucleosome structure was studied. Applications of spFRET microscopy of immobilized nucleosomes include analysis of their structural dynamics (DNA “breathing”), kinetics of formation/dissociation of DNA-protein complexes, formation and structure of stalled elongation complexes with RNAP, as well as conformational transitions in nucleosome structure in the course of transcription.
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U2 - 10.1007/978-3-319-46601-9_3
DO - 10.1007/978-3-319-46601-9_3
M3 - Conference contribution
SN - 9783319466002
T3 - Springer Proceedings in Physics
SP - 17
EP - 23
BT - 3rd International Multidisciplinary Microscopy and Microanalysis Congress (InterM) - Proceedings
A2 - Oral, Zehra Banu Bahsi
A2 - Oral, Ahmet Yavuz
PB - Springer Science and Business Media, LLC
Y2 - 19 October 2015 through 23 October 2015
ER -