Abstract
Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography.
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References
Kuhn CD, Geiger SR, Baumli S, Gartmann M, Gerber J, Jennebach S, Mielke T, Tschochner H, Beckmann R, Cramer P (2007) Functional architecture of RNA polymerase I. Cell 131(7):1260–1272. doi:10.1016/j.cell.2007.10.051
Roeder RG, Rutter WJ (1969) Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature 224(5216):234–237
Vannini A, Cramer P (2012) Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol Cell 45(4):439–446. doi:10.1016/j.molcel.2012.01.023
Warner JR (1999) The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24(11):437–440
Engel C, Sainsbury S, Cheung AC, Kostrewa D, Cramer P (2013) RNA polymerase I structure and transcription regulation. Nature 502(7473):650–655. doi:10.1038/nature12712
Fernandez-Tornero C, Moreno-Morcillo M, Rashid UJ, Taylor NM, Ruiz FM, Gruene T, Legrand P, Steuerwald U, Muller CW (2013) Crystal structure of the 14-subunit RNA polymerase I. Nature 502(7473):644–649. doi:10.1038/nature12636
Kostrewa D, Kuhn CD, Engel C, Cramer P (2015) An alternative RNA polymerase I structure reveals a dimer hinge. Acta Crystallogr D Biol Crystallogr 71(Pt 9):1850–1855. doi:10.1107/S1399004715012651
Geiger SR, Lorenzen K, Schreieck A, Hanecker P, Kostrewa D, Heck AJ, Cramer P (2010) RNA polymerase I contains a TFIIF-related DNA-binding subcomplex. Mol Cell 39(4):583–594. doi:10.1016/j.molcel.2010.07.028
Moreno-Morcillo M, Taylor NM, Gruene T, Legrand P, Rashid UJ, Ruiz FM, Steuerwald U, Muller CW, Fernandez-Tornero C (2014) Solving the RNA polymerase I structural puzzle. Acta Crystallogr D Biol Crystallogr 70(Pt 10):2570–2582. doi:10.1107/S1399004714015788
Appling FD, Schneider DA (2015) Purification of active RNA polymerase I from yeast. Methods Mol Biol 1276:281–289. doi:10.1007/978-1-4939-2392-2_16
Acknowledgements
I thank Patrick Cramer for supervising and supporting all research and method development. I acknowledge the contribution of all past and present members of the Pol I subgroup to a constant evolution of the protocol, especially Claus Kuhn and Stefan Jennebach. Thanks also to Carlo Bäjen, Tobias Gubbey, Sarah Sainsbury, Andrea Stutz and Youwei Xu. This work was funded by the Boehringer Ingelheim Fonds and the Max Planck Society.
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Engel, C. (2016). Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae . In: NĂ©meth, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_7
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DOI: https://doi.org/10.1007/978-1-4939-3792-9_7
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