Journal: Commun Biol / Year: 2022 Title: Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism. Authors: Emmanuel Saridakis / Rishi Vishwakarma / Josephine Lai-Kee-Him / Kevin Martin / Isabelle Simon / Martin Cohen-Gonsaud / Franck Coste / Patrick Bron / Emmanuel Margeat / Marc Boudvillain / Abstract: 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.
History
Deposition
Apr 1, 2021
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Header (metadata) release
Feb 23, 2022
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Map release
Feb 23, 2022
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Update
Feb 23, 2022
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Current status
Feb 23, 2022
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.9 Details: 20 mM Tris-Cl 5 % [v/v] glycerol 0.2 mM EDTA 0.2 M KCl 0.2 mM DTT pH 7.9
Grid
Model: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Details
Rho (0.5mg/mL) 10uM dC20 oligonucleotide 1mM ATP.
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Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 10888 / Average exposure time: 8.0 sec. / Average electron dose: 48.0 e/Å2 Details: The scheme of image recording was made with 7 images per position plus 1 middle, and as a series of 40 frames per movie.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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
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