E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

Abstract
Data availability
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Abstract

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ⁷⁰ region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Data availability

The cryo-EM density maps have been deposited in the EMDataBank under accession codes EMD-0348 [Eco TraR-Eσ70(I)], EMD-0349 [Eco TraR-Eσ70(II)], EMD-20231 [Eco TraR-Eσ70(III)], EMD-20230 (Eco Eσ70), EMD-20203 (rpsT P2-RPo), and EMD-20232 (rpsT P2-RPo2). The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6N57 [Eco TraR-Eσ70(I)], 6N58 [Eco TraR-Eσ70(II)], 6P1K (Eco Eσ70), and 6OUL (rpsT P2-RPo).

The following data sets were generated

1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli TraR-Esigma70(I)
EMDataResource, EMD-0348.

https://www.emdataresource.org/EMD-0348
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli TraR-Esigma70(II)
EMDataResource, EMD-0349.

https://www.emdataresource.org/EMD-0349
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli TraR-Esigma70(III)
EMDataResource, EMD-20231.

https://www.emdataresource.org/EMD-20231
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli Esigma70
EMDataResource, EMD-20230.

https://www.emdataresource.org/EMD-20230
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli Esigma70-rpsT P2 RPo(I)
EMDataResource, EMD-20203.

https://www.emdataresource.org/EMD-20203
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli Esigma70-rpsT P2 RPo(II)
EMDataResource, EMD-20232.

https://www.emdataresource.org/EMD-20232
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli TraR-Esigma70(I)
Protein Data Bank, 6N57.

http://www.rcsb.org/structure/6N57
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli TraR-Esigma70(II)
Protein Data Bank, 6N58.

http://www.rcsb.org/structure/6N58
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli Esigma70
Protein Data Bank, 6P1K.

http://www.rcsb.org/structure/6P1K
1. Chen J
2. Chiu C
3. Campbell EA
4. Darst SA
(2019) E. coli Esigma70-rpsT P2 RPo(I)
Protein Data Bank, 6OUL.

http://www.rcsb.org/structure/6OUL

The following previously published data sets were used

1. Bae B
2. Darst SA
(2013) Crystal structure analysis of the E. coli holoenzyme
Protein Data Bank, 4LJZ.

https://www.rcsb.org/structure/4LJZ
1. Bae B
2. Darst SA
(2013) Crystal structure analysis of the E. coli holoenzyme
Protein Data Bank, 4LK1.

https://www.rcsb.org/structure/4lk1
1. Murakami KS
2. Molodtsov V
(2017) X-ray crystal structure of Escherichia coli RNA polymerase and TraR complex
Protein Data Bank, 5W1S.

https://www.rcsb.org/structure/5W1S
1. Murakami KS
(2015) X-ray crystal structur of Escherichia coli RNA polymerase sigma70 holoenzyme
Protein Data Bank, 4YG2.

https://www.rcsb.org/structure/4YG2

Article and author information

Author details

James Chen

Laboratory of Molecular Biophysics, 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:" 0000-0002-2311-003X
Saumya Gopalkrishnan

Department of Bacteriology, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Courtney Chiu

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

Competing interests
The authors declare that no competing interests exist.
Albert Y Chen

Department of Bacteriology, University of Wisconsin-Madison, Madison, 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.
Richard L Gourse

Department of Bacteriology, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Wilma Ross

Department of Bacteriology, University of Wisconsin-Madison, Madison, 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 Institutes of Health (R01 GM114450)

Elizabeth A Campbell

National Institutes of Health (R01 GM37048)

Richard L Gourse

National Institutes of Health (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.