Structural basis for transcription complex disruption by the Mfd translocase

Abstract
Data availability
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Abstract

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

Data availability

The cryo-EM density maps have been deposited in the EMDataBank under accession codes EMD-21996 [L1(ATP)], EMD-22006 [L2(ADP)], EMD-22012 [I(ATP)], EMD-22039 [II(ATP)], EMD-22043 [III(ADP)], EMD-22044 [IV(ADP)], and EMD-22045 [V(ATP)]. The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6X26 [L1(ATP)], 6X2F [L2(ADP)], 6X2N [I(ATP)], 6X43 [II(ATP)], 6X4W [III(ADP)], 6XYY [IV(ADP)], and 6X5Q [V(ATP)].

The following data sets were generated

1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - L1 state
PDB, 6X26.

https://www.rcsb.org/structure/6X26
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - L1 state
EMDB, EMD-21996.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21996
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - L2 state
PDB, 6X2F.

https://www.rcsb.org/structure/6X2F
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - L2 state
EMDB, EMD-22006.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22006
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C1 state
PDB, 6X2N.

https://www.rcsb.org/structure/6X2N
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C1 state
EMDB, EMD-22012.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22012
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C2 state
PDB, 6X43.

https://www.rcsb.org/structure/6X43
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C2 state
EMDB, EMD-22039.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22039
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C3 state
PDB, 6X4W.

https://www.rcsb.org/structure/6X4W
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C3 state
EMDB, EMD-22043.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22043
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C4 state
PDB, 6XYY.

https://www.rcsb.org/structure/6XYY
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C4 state
EMDB, EMD-22044.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22044
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli RNA polymerase elongation complex - C5 state
PDB, 6X5Q.

https://www.rcsb.org/structure/6X5Q
1. Kang JY
2. Llewellyn E
3. Chen J
4. Darst SA
(2020) Mfd-bound E.coli aMfd-bound E.coli RNA polymerase elongation complex - C5 stateRNA polymerase elongation complex - L1 state
EMDB, EMD-22045.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-22045

The following previously published data sets were used

1. Deaconescu AM
2. Darst SA
(2006) Crystal structure of Escherichia coli transcription-repair coupling factor
PDB, 2EYQ.

https://www.rcsb.org/structure/2EYQ
1. Kang JY
2. Darst SA
(2017) CryoEM structure of crosslinked E.coli RNA polymerase elongation complex
PDB, 6ALF.

https://www.rcsb.org/structure/6ALF
1. Deaconescu AM
2. Grigorieff N
(2012) Core UvrA/TRCF complex
PDB, 4DFC.

https://www.rcsb.org/structure/4DFC
1. Kang JY
2. Darst SA
(2017) CryoEM structure of crosslinked E.coli RNA polymerase elongation complex
EMDB, EMD-8585.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8585/experiment

Article and author information

Author details

Jin Young Kang

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Eliza Llewellyn

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
James Chen

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Paul D B Olinares

Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3429-6618
Joshua Brewer

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Brian T Chait

Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Elizabeth A Campbell

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Seth A Darst

Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States

For correspondence
darst@rockefeller.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8241-3153

Funding

National Institute of General Medical Sciences (P41 GM109824)

Brian T Chait

National Institute of General Medical Sciences (P41 GM103314)

Brian T Chait

National Institute of General Medical Sciences (R01 GM114450)

Elizabeth A Campbell

National Institute of General Medical Sciences (R35 GM118130)

Seth A Darst

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.