ジャーナル: Nat Commun / 年: 2021 タイトル: Structural basis of RNA polymerase inhibition by viral and host factors. 著者: Simona Pilotto / Thomas Fouqueau / Natalya Lukoyanova / Carol Sheppard / Soizick Lucas-Staat / Luis Miguel Díaz-Santín / Dorota Matelska / David Prangishvili / Alan C M Cheung / Finn Werner / 要旨: RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution ...RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.
モデル: UltrAuFoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: GOLD / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 雰囲気: AIR 詳細: The grid was coated with graphene oxide prior to use
凍結
凍結剤: ETHANE / チャンバー内湿度: 94 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV
詳細
The sample was crosslinked with bis(sulfosuccinimidyl)suberate (BS3)
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: GATAN K3 (6k x 4k) / デジタル化 - サイズ - 横: 5760 pixel / デジタル化 - サイズ - 縦: 4092 pixel / 撮影したグリッド数: 1 / 実像数: 1676 / 平均電子線量: 44.46 e/Å2 詳細: Images were collected in movie-mode for a total of 40 frames in super-resolution mode and with a tilt of 30 degrees
Movie stacks were aligned and summed using MotionCor2
粒子像選択
選択した数: 1286432
CTF補正
ソフトウェア - 名称: Gctf 詳細: CTF aplitude was further corrected on selected particles after 3D recostruction to correct the 30 degrees tilt used during data collection
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.0)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.0)
最終 3次元分類
クラス数: 2 / ソフトウェア - 名称: RELION (ver. 3.0) 詳細: the particles were split 50% in each class providing 2 identical maps. The particles of both classes were pooled together prior 3D refinement and postprocessing.
FSC曲線 (解像度の算出)
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原子モデル構築 1
精密化
空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 57.7772 / 当てはまり具合の基準: CC
得られたモデル
PDB-7ok0: Cryo-EM structure of the Sulfolobus acidocaldarius RNA polymerase at 2.88 A