DNA-directed RNA polymerase II subunit RPB1 / General transcription and DNA repair factor IIH helicase subunit XPD/RAD3 / DNA-directed RNA polymerase II subunit RPB2 / TATA-box-binding protein / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 ...DNA-directed RNA polymerase II subunit RPB1 / General transcription and DNA repair factor IIH helicase subunit XPD/RAD3 / DNA-directed RNA polymerase II subunit RPB2 / TATA-box-binding protein / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB9 / Transcription initiation factor IIB / Mediator of RNA polymerase II transcription subunit 17 / Mediator of RNA polymerase II transcription subunit 22 / Mediator of RNA polymerase II transcription subunit 18 / Transcription initiation factor IIA large subunit / Transcription initiation factor IIA subunit 2 / Mediator of RNA polymerase II transcription subunit 9 / DNA-directed RNA polymerase II subunit RPB7 / Mediator of RNA polymerase II transcription subunit 20 / Transcription initiation factor TFIID subunit 14 / Transcription initiation factor IIE subunit alpha / Transcription initiation factor IIE subunit beta / Mediator of RNA polymerase II transcription subunit 8 / Mediator of RNA polymerase II transcription subunit 31 / Mediator of RNA polymerase II transcription subunit 6 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / Transcription initiation factor IIF subunit alpha / Transcription initiation factor IIF subunit beta / Mediator of RNA polymerase II transcription subunit 21 / General transcription and DNA repair factor IIH helicase/translocase subunit XPB/SSL2 / General transcription and DNA repair factor IIH subunit TFB2 / Mediator of RNA polymerase II transcription subunit 7 / Mediator of RNA polymerase II transcription subunit 4 / General transcription and DNA repair factor IIH subunit TFB5 / Mediator of RNA polymerase II transcription subunit 11 類似検索 - 構成要素
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
AI21144
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM36659
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM49985
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM103481
米国
National Science Foundation (NSF, United States)
1231306
米国
引用
ジャーナル: Cell / 年: 2016 タイトル: Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. 著者: Philip J Robinson / Michael J Trnka / David A Bushnell / Ralph E Davis / Pierre-Jean Mattei / Alma L Burlingame / Roger D Kornberg / 要旨: A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass ...A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 295 K / 装置: FEI VITROBOT MARK IV
詳細
For cryo-EM, samples were chemically stabilized by fixation in glycerol gradients with increasing concentrations of glutaraldehyde (0-0.1%). Peak gradient fractions (~0.2 mg/ml) containing glutaraldhyde were quenched upon the addition of 50 mM Tris-HCl (pH 7.5) before flash-freezing and storage under liquid nitrogen.
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電子顕微鏡法
顕微鏡
FEI TECNAI F20
撮影
フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) 平均電子線量: 15.0 e/Å2
試料ホルダーモデル: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER ホルダー冷却材: NITROGEN
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
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画像解析
CTF補正
ソフトウェア - 名称: CTFFIND (ver. 3)
初期モデル
モデルのタイプ: OTHER 詳細: Initial Model generated using EMAN 2.1 e2initialmodel.py program using a small subset of 2D class averages generated from Relion 1.4 Class2D.