Journal: Mol Cell / Year: 2021 Title: Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase. Authors: Brent De Wijngaert / Shemaila Sultana / Anupam Singh / Chhaya Dharia / Hans Vanbuel / Jiayu Shen / Daniel Vasilchuk / Sergio E Martinez / Eaazhisai Kandiah / Smita S Patel / Kalyan Das / Abstract: Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. ...Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
History
Deposition
Apr 10, 2020
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Header (metadata) release
Dec 9, 2020
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Map release
Dec 9, 2020
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Update
Oct 18, 2023
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Current status
Oct 18, 2023
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 8 K / Instrument: LEICA EM GP / Details: 5 uL sample; back blotting for 12 -14 second.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3500 / Average exposure time: 6.0 sec. / Average electron dose: 61.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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