RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB

Martin Overgaard, Jonas Borch, Kenn Gerdes

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2009-Sep-8
OriginalsprogEngelsk
TidsskriftJournal of Molecular Biology
Vol/bind394
Udgave nummer2
Sider (fra-til)183-196
Antal sider14
ISSN0022-2836
DOI
StatusUdgivet - 8. sep. 2009

Fingeraftryk

DNA
Protease La
Amino Acids
Messenger RNA
Printing
Transcriptional Activation
Proteolysis
Proteins

Citer dette

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title = "RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB",
abstract = "RelB, the Ribbon-Helix-Helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, forms a tight complex with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE - that is - RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes which map to important amino acid (aa) positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA-binding of a subset of these mutant RHH proteins using gel-shift assays, transcriptional fusion assays and a structure model of RelB-DNA revealed aa residues making crucial DNA-backbone contacts within the operator (relO) DNA. Mutational and foot printing analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6 bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon stimulated RelB degradation in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.",
author = "Martin Overgaard and Jonas Borch and Kenn Gerdes",
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RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB. / Overgaard, Martin; Borch, Jonas; Gerdes, Kenn.

I: Journal of Molecular Biology, Bind 394, Nr. 2, 08.09.2009, s. 183-196.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB

AU - Overgaard, Martin

AU - Borch, Jonas

AU - Gerdes, Kenn

PY - 2009/9/8

Y1 - 2009/9/8

N2 - RelB, the Ribbon-Helix-Helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, forms a tight complex with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE - that is - RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes which map to important amino acid (aa) positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA-binding of a subset of these mutant RHH proteins using gel-shift assays, transcriptional fusion assays and a structure model of RelB-DNA revealed aa residues making crucial DNA-backbone contacts within the operator (relO) DNA. Mutational and foot printing analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6 bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon stimulated RelB degradation in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.

AB - RelB, the Ribbon-Helix-Helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, forms a tight complex with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE - that is - RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes which map to important amino acid (aa) positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA-binding of a subset of these mutant RHH proteins using gel-shift assays, transcriptional fusion assays and a structure model of RelB-DNA revealed aa residues making crucial DNA-backbone contacts within the operator (relO) DNA. Mutational and foot printing analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6 bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon stimulated RelB degradation in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.

U2 - 10.1016/j.jmb.2009.09.006

DO - 10.1016/j.jmb.2009.09.006

M3 - Journal article

C2 - 19747491

VL - 394

SP - 183

EP - 196

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -