Abstract
A procedure was developed for the preparation of a stalled elongation complex of Escherichia coli RNA polymerase with the nucleosome for investigation by cryo-electron microscopy. We purified the complex from the excess of free RNA polymerase and unproductive complexes on heparin resin and concentrated it on an affine monolayer formed by lipids bound to Ni ions. The use of affinity grids with an immobilized lipid monolayer helps to prevent aggregation of the particles on the grid surface. This technique can be used in the future to obtain a three-dimensional reconstruction of the EC+39 elongation complex.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Dion, M.F., Kaplan, T., Kim, M., Buratowski, S., Friedman, N., and Rando, O.J., Dynamics of replication-independent histone turnover in budding yeast, Science, 2007, vol. 315, no. 5817, pp. 1405–1408.
Kireeva, M.L., Walter, W., Tchernajenko, V., Bondarenko, V., Kashlev, M., and Studitsky, V.M., Nucleosome remodeling induced by RNA polymerase II: Loss of the H2A/H2B dimer during transcription, Mol. Cell, 2002, vol. 9, no. 3, pp. 541–552.
Gaykalova, D.A., Kulaeva, O.I., Volokh, O., Shaytan, A.K., Hsieh, F.K., Kirpichnikov, M.P., Sokolova, O.S., and Studitsky, V.M., Structural analysis of nucleosomal barrier to transcription, Proc. Natl. Acad. Sci. U.S.A., 2015, vol. 112, no. 43, pp. 5787–5795.
Pestov, N.A., Gerasimova, N.S., Kulaeva, O.I., and Studitsky, V.M., Structure of transcribed chromatin is a sensor of DNA damage, Sci. Adv., 2015, vol. 1, no. 6.
Kulaeva, O.I., Gaykalova, D.A., Pestov, N.A., Golovastov, V.V., Vassylyev, D.G., Artsimovitch, I., and Studitsky, V.M., Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II, Nat. Struct. Mol. Biol., 2009, vol. 16, no. 12, pp. 1272–1278.
Murakami, K.S. and Darst, S.A., Bacterial RNA polymerases: The wholo story, Curr. Opin. Struct. Biol., 2003, vol. 13, pp. 31–39.
Luger, K., Mäder, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J., Crystal structure of the nucleosome core particle at 2.8 Å resolution, Nature, 1997, vol. 389, no. 6648, pp. 251–260.
Kudryashova, K.S., Nikitin, D.V., Chertkov, O.V., Gerasimova, N.S., Valieva, M.E., Studitsky, V.M., and Feofanov, A.V., Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy, Moscow Univ. Biol. Sci. Bull., 2015, vol. 70, no. 4, pp. 189–193.
Karlova, M.G., Volokh, O.I., Chertkov, O.V., Kirpichnikov, M.P., Studitsky, V.M., and Sokolova, O.S., Purification and concentration of RNA polymerase on Ni-lipid monolayers, Russ. J. Bioorg. Chem., 2017, vol. 43, no. 6, pp. 637–643.
Gaykalova, D.A., Kulaeva, O.I., Bondarenko, V.A., and Studitsky, V.M., Preparation and analysis of uniquely positioned mononucleosomes, Methods Mol. Biol., 2009, vol. 523, pp. 109–123.
Kelly, D.F., Abeyrathne, P.D., Dukovski, D., and Walz, T., The Affinity Grid: A pre-fabricated EM grid for monolayer purification, J. Mol. Biol., 2008, vol. 382, no. 2, pp. 423–433.
Walter, W., Kireeva, M.L., Studitsky, V.M., and Kashlev, M., Bacterial polymerase and yeast polymerase II use similar mechanisms for transcription through nucleosomes, J. Biol. Chem., 2003, vol. 278, no. 38, pp. 36148–36156.
Author information
Authors and Affiliations
Corresponding author
Additional information
Original Russian Text © O.V. Chertkov, M.G. Karlova, N.S. Gerasimova, O.S. Sokolova, 2018, published in Vestnik Moskovskogo Universiteta, Seriya 16: Biologiya, 2018, Vol. 73, No. 3, pp. 173–177.
The article was translated by the authors.
About this article
Cite this article
Chertkov, O.V., Karlova, M.G., Gerasimova, N.S. et al. Purification of RNA Polymerase Elongation Complexes for Cryoelectron Microscopy Investigation. Moscow Univ. Biol.Sci. Bull. 73, 142–145 (2018). https://doi.org/10.3103/S0096392518030045
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.3103/S0096392518030045