Volume 6 Supplement 1

Epigenetics and Chromatin: Interactions and processes

Open Access

Pausing as a mechanism of nucleosome recovery

  • Han-Wen Chang1,
  • Olga I Kulaeva1, 2,
  • Alexey Shaytan2,
  • M Kibanov2,
  • K Severinov3,
  • David J Clark4 and
  • Vasily M Studitsky1, 2
Epigenetics & Chromatin20136(Suppl 1):P14

DOI: 10.1186/1756-8935-6-S1-P14

Published: 18 March 2013

Background

Nucleosome survival during transcription is important for the maintenance of chromatin integrity, gene regulation, cell survival, and aging. In according to the studies in vitro and in vivo, Pol II pauses at positions inside of a nucleosome and nucleosome survives after Pol II transcription through chromatin [1]. A key intermediate, Ø-loop (EC+49), formed at +45 region inside of nucleosome and Pol II pausing at +45 nucleosomal region positively correlated to nucleosome survival [2, 3]. A putative electrostatic interaction between Pol II and histone octamer has also been predicted to stabilize the Ø-loop intermediate by structural modeling[3]. In this study, we will provide more evidences to link those characteristics together.

Materials and methods

Protein purification [3][4]

Computational structure modeling [3]

Transcription assay, Nucleosome fate and DNase I footprinting [3, 5]

Results

In structural modeling, three negatively charged surfaces (regions 1, 2, 3) were identified on Rpb1, a large subunit of yeast Pol II, and plausibly interacted to histone octamer, especially H2B N-tail region, by the electrostatic force. Results of transcriptions by Thermus thermophilus (T.th.) and Thermus aquaticus (T.aq.) RNAPs which contained less net negative charges at all three regions showed lower +45 pausing, no Ø-loop formation and nucleosome displacement. Finally, lower nucleosomal barrier was also shown during the transcription by mutated yeast Pol II which contains less negatively charged at region 2 of Rpb1.

Conclusions

Collectively, we demonstrated the positive correlations of higher negative net charge of the interacting region of RNAP, the stronger +45 barrier, more efficient nucleosome survival and more efficient Ø-loop intermediate formation during transcription.

Authors’ Affiliations

(1)
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, and Faculty of Biology
(2)
Moscow State University
(3)
Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey
(4)
Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health

References

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Copyright

© Chang et al; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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