National Institutes of Health/National Institute of General Medical Sciences
R35 GM118130
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2019 タイトル: Structural basis for transcription activation by Crl through tethering of σ and RNA polymerase. 著者: Alexis Jaramillo Cartagena / Amy B Banta / Nikhil Sathyan / Wilma Ross / Richard L Gourse / Elizabeth A Campbell / Seth A Darst / 要旨: In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional ...In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In , , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ-regulon by promoting the association of σ with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ-RNAP in an open promoter complex with a σ-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ (σ), the structure, along with -benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β'-subunit that we call the β'-clamp-toe (β'CT). Deletion of the β'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β'CT interaction. We conclude that Crl activates σ-dependent transcription in part through stabilizing σ-RNAP by tethering σ and the β'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 298.15 K / 装置: FEI VITROBOT MARK IV
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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 K2 SUMMIT (4k x 4k) 平均電子線量: 1.42 e/Å2
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 倍率(公称値): 22500
試料ステージ
ホルダー冷却材: NITROGEN
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
粒子像選択
選択した数: 658000
CTF補正
ソフトウェア - 名称: RELION (ver. 3)
初期モデル
モデルのタイプ: INSILICO MODEL In silico モデル: Alpha, beta, beta', and omega subunits from PDB: 5VT0. SigmaS subunit from 5IPL. Crl from 3RPJ. Mostly de novo built DNA.