ジャーナル: Commun Biol / 年: 2022 タイトル: Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism. 著者: Emmanuel Saridakis / Rishi Vishwakarma / Josephine Lai-Kee-Him / Kevin Martin / Isabelle Simon / Martin Cohen-Gonsaud / Franck Coste / Patrick Bron / Emmanuel Margeat / Marc Boudvillain / 要旨: The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis ...The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [Rho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of Rho at 3.3 Å resolution. The Rho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of Rho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of Rho and provides a framework for future antibiotic development.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 295.15 K / 装置: FEI VITROBOT MARK IV
詳細
Rho (0.5mg/mL) 10uM dC20 oligonucleotide 1mM ATP.
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電子顕微鏡法
顕微鏡
TFS KRIOS
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - 画像ごとのフレーム数: 1-40 / 撮影したグリッド数: 1 / 実像数: 10888 / 平均露光時間: 8.0 sec. / 平均電子線量: 48.0 e/Å2 詳細: The scheme of image recording was made with 7 images per position plus 1 middle, and as a series of 40 frames per movie.
K2 Summitdirect electron detector configured in counting mode, mounted on a Gatan Quantum 967 LS energy filter using a 20 eV slit width in zero loss mode