Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus

Marina N. Vassylyeva, Jookyung Lee, Shun Ichi Sekine, Oleg Laptenko, Seiki Kuramitsu, Takehiko Shibata, Yorinao Inoue, Sergei Borukhov, Dmitry G. Vassylyev, Shigeyuki Yokoyama

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Abstract

RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (α2, β, β′, ω and σ70) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 Å resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P32, with unit-cell parameters a = b = 236.35, c = 249.04 Å, and have perfect twinning along the threefold axis. A complete data set at 3 Å resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the σ70 subunit is now in progress.

Original languageEnglish (US)
Pages (from-to)1497-1500
Number of pages4
JournalActa Crystallographica Section D: Biological Crystallography
Volume58
Issue number9
DOIs
StatePublished - Sep 1 2002

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All Science Journal Classification (ASJC) codes

  • Structural Biology

Cite this

Vassylyeva, M. N., Lee, J., Sekine, S. I., Laptenko, O., Kuramitsu, S., Shibata, T., Inoue, Y., Borukhov, S., Vassylyev, D. G., & Yokoyama, S. (2002). Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus. Acta Crystallographica Section D: Biological Crystallography, 58(9), 1497-1500. https://doi.org/10.1107/S0907444902011770