Пробоподготовка элонгационного комплекса +39 для криоэлектронной микроскопии

Транскрипция в клетке осуществляется специализированными ферментами – РНК-полимеразами. РНК-полимеразы транскрибируют ДНК с образованием элонгационных комплексов (ЭК), в том числе участвующих в регуляции транскрипции. Криоэлектронная микроскопия (криоЭМ) позволяет определять структуры этих комплексов и выяснять механизмы транскрипции хроматина. Однако подготовка образцов ЭК, пригодных для изучения методом криоЭМ, представляет определенные сложности. В данной работе проведена разработка протоколов для подготовки образов ЭК с положением активного центра РНКП в позиции +39 от входа в нуклеосому (ЭК+39). Образование комплексов подтверждалось методами электрофореза и электронной микроскопии негативного контрастирования. Разработанные методы могут быть использованы для изучения ЭК+39 методом криоЭМ.

1. Landick R. The regulatory roles and mechanism of transcriptional pausing. Biochem. Soc. Trans. 2006;34(Pt. 6):1062–1066.

2. Farnung L., Vos S.M., Cramer P. Structure of transcribing RNA polymerase II-nucleosome complex. Nat. Commun. 2018;9(1):5432.

3. Kujirai T., Ehara H., Fujino Y., Shirouzu M., Sekine S.I., Kurumizaka H. Structural basis of the nucleosome transition during RNA polymerase II passage. Science. 2018;362(6414):595–598.

4. Ehara H., Kujirai T., Shirouzu M., Kurumizaka H., Sekine S. ichi. Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT. Science. 2022;377(6611):eabp9466.

5. Farnung L., Ochmann M., Garg G., Vos S.M., Cramer P. Structure of a backtracked hexasomal intermediate of nucleosome transcription. Mol. Cell. 2022;82(17):3126-3134.e7.

6. Osumi K., Kujirai T., Ehara H., Ogasawara M., Kinoshita C., Saotome M., Kagawa W., Sekine S.I., Takizawa Y., Kurumizaka H. Structural basis of damaged nucleotide recognition by transcribing RNA polymerase II in the nucleosome. J. Mol. Biol. 2023;435(13):168130.

7. Naganuma M., Kujirai T., Ehara H., Uejima T., Ito T., Goto M., Aoki M., Henmi M., Miyamoto-Kohno S., Shirouzu M., Kurumizaka H., Sekine S. ichi. Structural insights into promoter-proximal pausing of RNA polymerase II at +1 nucleosome. Sci. Adv. 2025;11(10):eadu0577.

8. Kujirai T., Kato J., Yamamoto K., Hirai S., Fujii T., Maehara K., Harada A., Negishi L., Ogasawara M., Yamaguchi Y., Ohkawa Y., Takizawa Y., Kurumizaka H. Multiple structures of RNA polymerase II isolated from human nuclei by ChIP-CryoEM analysis. Nat. Commun. 2025;16(1):4724.

9. Kulaeva O.I., Gaykalova D.A., Pestov N.A., Golovastov V.V., Vassylyev D.G., Artsimovitch I., Studitsky V.M. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat. Struct. Mol. Biol. 2009;16(12):1272–1278.

10. Walter W., Kireeva M.L., Studitsky V.M., Kashlev M. Bacterial polymerase and yeast polymerase II use similar mechanisms for transcription through nucleosomes. J. Biol. Chem. 2003;278(38):36148–36156.

11. Gaykalova D.A., Kulaeva O.I., Pestov N.A., Hsieh F.K., Studitsky V.M. Experimental analysis of the mechanism of chromatin remodeling by RNA polymerase II. Nucleosomes, Histones and Chromatin Part A. Methods in Enzymology, vol. 512. Eds. C. Wu and C.D. Allis. Academic Press; 2012:293–314.

12. Saba J., Chua X.Y., Mishanina T.V., Nayak D., Windgassen T.A., Mooney R.A., Landick R. The elemental mechanism of transcriptional pausing. eLife. 2019;8:e40981.

13. Song E., Hwang S., Munasingha P.R., Seo Y.S., Kang J.Y., Kang C., Hohng S. Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination. Nucleic Acids Res. 2023;51(6):2778–2789.

14. Gromak N., West S., Proudfoot N.J. Pause sites promote transcriptional termination of mammalian RNA polymerase II. Mol. Cell. Biol. 2006;26(10):3986–3996.

15. Stoeger T., Grant R.A., McQuattie-Pimentel A.C., et al. Aging is associated with a systemic length-associated transcriptome imbalance. Nat. Aging. 2022;2(12):1191–1206.

16. Modur V., Singh N., Mohanty V., et al. Defective transcription elongation in a subset of cancers confers immunotherapy resistance. Nat. Commun. 2018;9(1):4410.

17. Chang H.W., Kulaeva O.I., Shaytan A.K., Kibanov M., Kuznedelov K., Severinov K.V., Kirpichnikov M.P., Clark D.J., Studitsky V.M. Analysis of the mechanism of nucleosome survival during transcription. Nucleic Acids Res. 2014;42(3):1619–1627.

18. Artsimovitch I., Svetlov V., Murakami K.S., Landick R. Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions. J. Biol. Chem. 2003;278(14):12344–12355.

19. Luger K., Rechsteiner T.J., Richmond T.J. Preparation of nucleosome core particle from recombinant histones. Chromatin. Methods in Enzymology, vol. 304. Eds. P.M. Wassarman and A.P. Wolffe. Academic Press.1999;304:3–19.

20. Klinker H., Haas C., Harrer N., Becker P.B., Mueller-Planitz F. Rapid purification of recombinant histones. PLoS One. 2014;9(8):e104029.

21. Mastronarde D.N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 2005;152(1):36–51.

22. Scheres S.H.W. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 2012;180(3):519–530.

23. Chertkov O.V., Karlova M.G., Gerasimova N.S., Sokolova O.S. Purification of RNA polymerase elongation complexes for cryoelectron microscopy investigation. Mosc. Univ. Biol. Sci. Bull. 2018;73(3):142–145.

24. Kubori T., Shimamoto N. A branched pathway in the early stage of transcription byEscherichia coliRNA polymerase. J. Mol. Biol. 1996;256(3):449–457.

25. Plaskon D.M., Henderson K.L., Felth L.C., Molzahn C.M., Evensen C., Dyke S., Shkel I.A., Record M.T. Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise. Proc. Natl. Acad. Sci. U.S.A. 2021;118(30):e2021941118.

26. Chertkov O.V., Karlova M.G., Studitsky V.M., Sokolova O.S. The three-dimensional structure of (+39) RNA-polymerase elongation complex determined by cryo-electron microscopy. Microsc. Microanal. 2019;25(S2):1332–1333.