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Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

König B, Müller JJ, Lanka E, Heinemann U - Nucleic Acids Res. (2009)

Bottom Line: As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site.KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB.We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

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Specific binding of KorA to its operator-binding site. (A) Specific KorA-OA* contacts in the crystal. Arg48 and Gln53 of the recognition helix (α4) of one KorA monomer form specific hydrogen bonds to G4, T7, C10 and T11, in one half-site of the symmetric operator sequence. (B) In vitro binding assay of purified wt KorA and mutants binding to DNA. The 436-bp fragment carries the class I OA site (korAp) of plasmid RP4, the other fragments serve as competitor DNA; 7.5 pmol of wt and each mutant protein were applied. The DNA mixture contained 0.15 pmol of each fragment. OA, DNA fragment containing OA; OA°, complex of OA with wt KorA; M, 100-bp DNA ladder. (C) Calorimetric titration of KorA with 18-bp OA. The panel shows the integrated heat released after correction of dilution (data points, squares) and the curve of best fit (red) for one KorA dimer binding to a single OA site.
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Figure 5: Specific binding of KorA to its operator-binding site. (A) Specific KorA-OA* contacts in the crystal. Arg48 and Gln53 of the recognition helix (α4) of one KorA monomer form specific hydrogen bonds to G4, T7, C10 and T11, in one half-site of the symmetric operator sequence. (B) In vitro binding assay of purified wt KorA and mutants binding to DNA. The 436-bp fragment carries the class I OA site (korAp) of plasmid RP4, the other fragments serve as competitor DNA; 7.5 pmol of wt and each mutant protein were applied. The DNA mixture contained 0.15 pmol of each fragment. OA, DNA fragment containing OA; OA°, complex of OA with wt KorA; M, 100-bp DNA ladder. (C) Calorimetric titration of KorA with 18-bp OA. The panel shows the integrated heat released after correction of dilution (data points, squares) and the curve of best fit (red) for one KorA dimer binding to a single OA site.

Mentions: For ITC experiments, purified KorA and 18-bp oligonucleotides (OA, 5′-CTT GTT TAG CTA AAC ATT-3′ and 5′-AAT GTT TAG CTA AAC AAG-3′), were dialyzed against the buffer, 20 mM Tris/HCl pH 8, 100 mM NaCl and 3 mM MgCl2. Samples were degassed for ∼5 min by vacuum aspiration prior to loading, and all titrations were carried out at 310 K. Experiments were performed on a VP MicroCal VP-ITC titration calorimeter (MicroCal Inc., Northampton, MA) using the VPViewer 2000 software for instrument control and data acquisition. KorA solution measuring 40 µM was filled in a stirred (310 r.p.m.) reaction cell of 1.4 ml. Injections, each of 10-µl volume (first injection always 5-µl volume) and 10-s duration with a 4-min interval between injections, were carried out using a syringe filled with 200 µM 18-mer OA (Figure 5C) solution. Thermogram analysis was performed using the software ORIGIN (version 7.0) provided by the manufacturer.


Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation.

König B, Müller JJ, Lanka E, Heinemann U - Nucleic Acids Res. (2009)

Specific binding of KorA to its operator-binding site. (A) Specific KorA-OA* contacts in the crystal. Arg48 and Gln53 of the recognition helix (α4) of one KorA monomer form specific hydrogen bonds to G4, T7, C10 and T11, in one half-site of the symmetric operator sequence. (B) In vitro binding assay of purified wt KorA and mutants binding to DNA. The 436-bp fragment carries the class I OA site (korAp) of plasmid RP4, the other fragments serve as competitor DNA; 7.5 pmol of wt and each mutant protein were applied. The DNA mixture contained 0.15 pmol of each fragment. OA, DNA fragment containing OA; OA°, complex of OA with wt KorA; M, 100-bp DNA ladder. (C) Calorimetric titration of KorA with 18-bp OA. The panel shows the integrated heat released after correction of dilution (data points, squares) and the curve of best fit (red) for one KorA dimer binding to a single OA site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2665229&req=5

Figure 5: Specific binding of KorA to its operator-binding site. (A) Specific KorA-OA* contacts in the crystal. Arg48 and Gln53 of the recognition helix (α4) of one KorA monomer form specific hydrogen bonds to G4, T7, C10 and T11, in one half-site of the symmetric operator sequence. (B) In vitro binding assay of purified wt KorA and mutants binding to DNA. The 436-bp fragment carries the class I OA site (korAp) of plasmid RP4, the other fragments serve as competitor DNA; 7.5 pmol of wt and each mutant protein were applied. The DNA mixture contained 0.15 pmol of each fragment. OA, DNA fragment containing OA; OA°, complex of OA with wt KorA; M, 100-bp DNA ladder. (C) Calorimetric titration of KorA with 18-bp OA. The panel shows the integrated heat released after correction of dilution (data points, squares) and the curve of best fit (red) for one KorA dimer binding to a single OA site.
Mentions: For ITC experiments, purified KorA and 18-bp oligonucleotides (OA, 5′-CTT GTT TAG CTA AAC ATT-3′ and 5′-AAT GTT TAG CTA AAC AAG-3′), were dialyzed against the buffer, 20 mM Tris/HCl pH 8, 100 mM NaCl and 3 mM MgCl2. Samples were degassed for ∼5 min by vacuum aspiration prior to loading, and all titrations were carried out at 310 K. Experiments were performed on a VP MicroCal VP-ITC titration calorimeter (MicroCal Inc., Northampton, MA) using the VPViewer 2000 software for instrument control and data acquisition. KorA solution measuring 40 µM was filled in a stirred (310 r.p.m.) reaction cell of 1.4 ml. Injections, each of 10-µl volume (first injection always 5-µl volume) and 10-s duration with a 4-min interval between injections, were carried out using a syringe filled with 200 µM 18-mer OA (Figure 5C) solution. Thermogram analysis was performed using the software ORIGIN (version 7.0) provided by the manufacturer.

Bottom Line: As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site.KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB.We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-A resolution. KorA is present as a symmetric dimer and contacts DNA via a helix-turn-helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

Show MeSH
Related in: MedlinePlus