Structural basis of transcription arrest by coliphage HK022 nun in an Escherichia coli RNA polymerase elongation complex

Abstract
Data availability
Article and author information
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Abstract

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λΔNA.To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic-acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

Data availability

The following data sets were generated

1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of HK022 Nun - E. coli RNA polymerase elongation complex
Publicly available at the RCSB Protein Data Bank (accession no: 5UP6).

http://www.rcsb.org/pdb/explore/explore.do?structureId=5UP6
1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of HK022 Nun - E. coli RNA polymerase elongation complex
Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8584).

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8584
1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of crosslinked E.coli RNA polymerase elongation complex
Publicly available at the RCSB Protein Data Bank (accession no: 5UPA).

http://www.rcsb.org/pdb/explore/explore.do?structureId=5UPA
1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of crosslinked E.coli RNA polymerase elongation complex
Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8585).

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8585
1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of E.coli RNA polymerase elongation complex
Publicly available at the RCSB Protein Data Bank (accession no: 5UPC).

http://www.rcsb.org/pdb/explore/explore.do?structureId=5UPC
1. Jin Young Kang
2. Seth A. Darst
(2017) CryoEM structure of E.coli RNA polymerase elongation complex
Publicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8586).

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8586

The following previously published data sets were used

1. Bae B
2. Darst SA
(2013) Crystal Structure Analysis of the E.coli holoenzyme
Publicly available at the RCSB Protein Data Bank (accession no: 4LJZ).

http://www.rcsb.org/pdb/explore/explore.do?structureId=4LJZ
1. Vassylyev DG
2. Vassylyeva MN
(2007) Crystal structure of the T. thermophilus RNAP polymerase elongation complex with the NTP substrate analog
Publicly available at the RCSB Protein Data Bank (accession no: 2O5J).

http://www.rcsb.org/pdb/explore/explore.do?structureId=2O5J

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.
Paul Dominic 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.
James Chen

Laboratory of Molecular Biophysics, 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.
Arkady Mustaev

Public Health Research Institute, Newark, 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.
Max E Gottesman

Department of Microbiology and Immunology, Columbia University Medical Center, 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 Institutes of Health (R35 GM118130)

Seth A Darst

National Institutes of Health (R01 GM037219)

Max E Gottesman

National Institutes of Health (P41 GM103314)

Brian T Chait

Public Health Research Institute Research Support grant

Arkady Mustaev

National Institutes of Health (P41 GM109824)

Brian T Chait

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.