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Title: Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB

Abstract

RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379–405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204–209, 2010,https://doi.org/10.1016/j.mib.2010.01.002; D. J. Clarke, Future Microbiol 5:1173–1184, 2010,https://doi.org/10.2217/fmb.10.83). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6–17, 2012,https://doi.org/10.1094/MPMI-08-11-0207). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allowsmore » RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria.« less

Authors:
 [1];  [2];  [3];  [2];  [4];  [1]
  1. Northwestern University Feinberg School of Medicine, Chicago, IL (United States)
  2. Loyola University Chicago, Stritch School of Medicine, Maywood, IL (United States)
  3. Northwestern Univ., Evanston, IL (United States)
  4. Center for Structural Genomics of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Illinois at Urbana-Champaign, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); NIAID; National Institutes of Health (NIH); HHS
OSTI Identifier:
1493948
Alternate Identifier(s):
OSTI ID: 1499720
Grant/Contract Number:  
SC0012443; AC02-06CH11357; NIH R01AI083640; NIH 2R01AI083640-06A1; R01AI083640; 2R01AI083640-06A1
Resource Type:
Published Article
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Name: mBio (Online); Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Filippova, Ekaterina V., Zemaitaitis, Bozena, Aung, Theint, Wolfe, Alan J., Anderson, Wayne F., and Hendrickson, Wayne A. Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB. United States: N. p., 2018. Web. doi:10.1128/mBio.01993-17.
Filippova, Ekaterina V., Zemaitaitis, Bozena, Aung, Theint, Wolfe, Alan J., Anderson, Wayne F., & Hendrickson, Wayne A. Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB. United States. doi:10.1128/mBio.01993-17.
Filippova, Ekaterina V., Zemaitaitis, Bozena, Aung, Theint, Wolfe, Alan J., Anderson, Wayne F., and Hendrickson, Wayne A. Tue . "Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB". United States. doi:10.1128/mBio.01993-17.
@article{osti_1493948,
title = {Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB},
author = {Filippova, Ekaterina V. and Zemaitaitis, Bozena and Aung, Theint and Wolfe, Alan J. and Anderson, Wayne F. and Hendrickson, Wayne A.},
abstractNote = {RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379–405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204–209, 2010,https://doi.org/10.1016/j.mib.2010.01.002; D. J. Clarke, Future Microbiol 5:1173–1184, 2010,https://doi.org/10.2217/fmb.10.83). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6–17, 2012,https://doi.org/10.1094/MPMI-08-11-0207). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allows RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria.},
doi = {10.1128/mBio.01993-17},
journal = {mBio (Online)},
number = 1,
volume = 9,
place = {United States},
year = {2018},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1128/mBio.01993-17

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Cited by: 2 works
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Figures / Tables:

FIG 1 FIG 1: Oligomeric state and DNA binding activity of RcsB. (A) EMSA of RcsB against DNA22. Lane 1, free DNA; lane 2, DNA in the presence of RcsB; lane 3, DNA in the presence of RcsB and carbamoyl phosphorylate (CPh); lane 4, DNA in the presence of RcsB and phosphoramidemore » (PA); lane 5, DNA in the presence of RcsB, CPh, and unlabeled mutated (mut) DNA22; lane 6, DNA in the presence of RcsB, CPh, and unlabeled (unlab) wild-type DNA22. HMM complex, higher-molecularmass complex. (B) The SEC-MALS elution profiles of RcsB and RcsB in the presence of phosphodonor (RcsB*). The horizontal bold lines show the determined molecular masses (MM). The theoretical MM of the RcsB monomer is 24 kDa. (C) The SEC-MALS elution profiles of RcsB and RcsB* (with phosphodonor) in the presence of DNA22. The horizontal bold lines show the calculated MM of the RcsB-DNA complex. The theoretical MM of the RcsB dimer bound to the 22-bp DNA duplex is 63 kDa. The second peak eluted around 31 min corresponds to unbound DNA. (D and E) SPR sensograms (colored curves) of unphosphorylated and carbamoyl-phosphorylated RcsB and DNA22, respectively. KD values were calculated based on a 1:1 kinetic model, in which one RcsB dimer interacts with one double-stranded DNA. The fitting curves are shown in black. The corresponding concentrations of RcsB are indicated above the SPR curves. (F) RcsB-binding site from the flhDC promoter and DNA22 and DNA18 sequences.« less

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