Biotechnology and Biological Sciences Research Council (BBSRC)
BB/M011186/1
United Kingdom
Citation
Journal: Nature / Year: 2024 Title: Translation selectively destroys non-functional transcription complexes. Authors: Jason Woodgate / Hamed Mosaei / Pavel Brazda / Flint Stevenson-Jones / Nikolay Zenkin / Abstract: Transcription elongation stalls at lesions in the DNA template. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site. Here we ...Transcription elongation stalls at lesions in the DNA template. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site. Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.
Name: mRNA / type: rna / ID: 7 / Number of copies: 1
Source (natural)
Organism: Escherichia coli (E. coli)
Molecular weight
Theoretical: 6.149746 KDa
Sequence
String:
AAUAAAAUCG AGCGUGUGA
+
Macromolecule #8: ZINC ION
Macromolecule
Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
+
Macromolecule #9: MAGNESIUM ION
Macromolecule
Name: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
-
Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
1 mg/mL
Buffer
pH: 7.4
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 250 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.00026000000000000003 kPa / Details: Positive Charge
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
TFS GLACIOS
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 16264 / Average exposure time: 3.2 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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