Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA

Abstract
Data availability
Article and author information
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Abstract

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish thatRbpA and CarD cooperatively stimulate formation of anintermediate (RP2) leading to RPo; formation of RP2 islikely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2.We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the -10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcriptionby RbpA and CarD.

Data availability

The following data sets were generated

(2016) Protein Data Bank (PDB)
5TW1.

http://www.rcsb.org/pdb/home/home.do
1. A. Fay
2. M.S. Glickman
(2016) Gene Expression Omnibus (GEO)
GSE89773.

https://www.ncbi.nlm.nih.gov/geo/

The following previously published data sets were used

(2014) Protein Data Bank (PDB)
4X8K.

http://www.rcsb.org/pdb/home/home.do
1. Bortoluzzi
2. A.
3. Muskett
4. F.W.
5. Waters
6. L.C.
7. Addis
8. P.W.
9. Rieck
10. B.
11. Munder
12. T.
13. Schleier
14. S.
15. Forti
16. F.
17. Ghisotti
18. D.
19. Carr
20. M.D.
21. O'Hare
22. H.M.
(2013) Protein Data Bank (PDB)
2M4V.

http://www.rcsb.org/pdb/home/home.do
1. Bae
2. B.
3. Darst
4. S.A.
(2015) Protein Data Bank (PDB)
4XLR.

http://www.rcsb.org/pdb/home/home.do
1. Bae
2. B.
3. Darst
4. S.A.
(2015) Protein Data Bank (PDB)
4XLP.

http://www.rcsb.org/pdb/home/home.do

Article and author information

Author details

Elizabeth A Hubin

The Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Allison Fay

Immunology Program, Sloan-Kettering Institute, New York, United States

Competing interests
The authors declare that no competing interests exist.
Catherine Xu

Department of Chemistry, University of Cambridge, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
James M Bean

Immunology Program, Sloan-Kettering Institute, New York, United States

Competing interests
The authors declare that no competing interests exist.
Ruth M Saecker

Department of Chemistry, University of Cambridge, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
Michael S Glickman

Immunology Program, Sloan-Kettering Institute, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7918-5164
Seth A Darst

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
Elizabeth A Campbell

The Rockefeller University, New York, United States

For correspondence
elizabeth.campbell0@gmail.com

Competing interests
The authors declare that no competing interests exist.

Funding

National Institutes of Health (RO1 GM114450)

Elizabeth A Campbell

National Institutes of Health (P30 CA008748)

Michael S Glickman

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.