Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain

Wei Shi; Wei Zhou; Baoyue Zhang; Shaojia Huang; Yanan Jiang; Abigail Schammel; Yangbo Hu; Bin Liu

doi:10.15252/embj.2020104389

Structural basis of bacterial σ²⁸-mediated transcription reveals roles of the RNA polymerase zinc-binding domain

Wei Shi, Wei Zhou, Baoyue Zhang, Shaojia Huang, Yanan Jiang, Abigail Schammel, Yangbo Hu, Bin Liu

The Hormel Institute

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

Abstract

In bacteria, σ²⁸ is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ²⁸-dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ²⁸-dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ²⁸-RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ⁷⁰ family factors to enhance transcription from promoters with weak σ4/−35 element interactions.

Original language	English (US)
Article number	e104389
Journal	EMBO Journal
Volume	39
Issue number	14
DOIs	https://doi.org/10.15252/embj.2020104389
State	Published - Jul 15 2020

Bibliographical note

Funding Information:
The cryo-EM data were collected at the cryo-electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start-up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged.

Funding Information:
The cryo‐EM data were collected at the cryo‐electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start‐up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged.

Publisher Copyright:
© 2020 The Authors

Keywords

Cryo-EM
ZBD relocation
flagellar gene regulation
transcription initiation complex
σ

Publisher link

10.15252/embj.2020104389

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@article{9ed72246b03f4fe3a11d96b8cd4a1759,

title = "Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain",

abstract = "In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28-dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28-dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28-RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/−35 element interactions.",

keywords = "Cryo-EM, ZBD relocation, flagellar gene regulation, transcription initiation complex, σ",

author = "Wei Shi and Wei Zhou and Baoyue Zhang and Shaojia Huang and Yanan Jiang and Abigail Schammel and Yangbo Hu and Bin Liu",

note = "Funding Information: The cryo-EM data were collected at the cryo-electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start-up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged. Funding Information: The cryo‐EM data were collected at the cryo‐electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start‐up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged. Publisher Copyright: {\textcopyright} 2020 The Authors",

year = "2020",

month = jul,

day = "15",

doi = "10.15252/embj.2020104389",

language = "English (US)",

volume = "39",

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T1 - Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain

AU - Shi, Wei

AU - Zhou, Wei

AU - Zhang, Baoyue

AU - Huang, Shaojia

AU - Jiang, Yanan

AU - Schammel, Abigail

AU - Hu, Yangbo

AU - Liu, Bin

N1 - Funding Information: The cryo-EM data were collected at the cryo-electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start-up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged. Funding Information: The cryo‐EM data were collected at the cryo‐electron microscopy facility in the Hormel Institute, University of Minnesota, which is funded by the Hormel Foundation. This work was supported by the start‐up funding granted to B.L. from the Hormel Institute, University of Minnesota, and the National Natural Science Foundation of China to Y.H. (#31670134). Support from the Youth Innovation Promotion Association, CAS, to Y.H. and help from the Core Facility and Technical Support of Wuhan Institute of Virology in radioactive and fluorescent tests are also acknowledged. Publisher Copyright: © 2020 The Authors

PY - 2020/7/15

Y1 - 2020/7/15

N2 - In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28-dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28-dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28-RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/−35 element interactions.

AB - In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28-dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28-dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28-RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/−35 element interactions.

KW - Cryo-EM

KW - ZBD relocation

KW - flagellar gene regulation

KW - transcription initiation complex

KW - σ

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