Journal: Mol Cell / Year: 2019 Title: Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor. Authors: Ferdinand Krupp / Nelly Said / Yong-Heng Huang / Bernhard Loll / Jörg Bürger / Thorsten Mielke / Christian M T Spahn / Markus C Wahl / Abstract: Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo- ...Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the β subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.
History
Deposition
May 30, 2018
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Header (metadata) release
Aug 22, 2018
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Map release
Feb 20, 2019
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Update
Apr 17, 2019
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Current status
Apr 17, 2019
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: Quantifoil R2/4 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER Details: A thin carbon support film was placed over the holey carbon film.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK II
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 20.0 sec. / Average electron dose: 69.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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