Limits...
Repressor CopG prevents access of RNA polymerase to promoter and actively dissociates open complexes.

Hernández-Arriaga AM, Rubio-Lepe TS, Espinosa M, del Solar G - Nucleic Acids Res. (2009)

Bottom Line: First, CopG hindered binding of RNA polymerase to the promoter.Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr).A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Replication of the promiscuous plasmid pMV158 requires expression of the initiator repB gene, which is controlled by the repressor CopG. Genes repB and copG are co-transcribed from promoter P(cr). We have studied the interactions between RNA polymerase, CopG and the promoter to elucidate the mechanism of repression by CopG. Complexes formed at 0 degrees C and at 37 degrees C between RNA polymerase and P(cr) differed from each other in stability and in the extent of the DNA contacted. The 37 degrees C complex was very stable (half-life of about 3 h), and shared features with typical open complexes generated at a variety of promoters. CopG protein repressed transcription from P(cr) at two different stages in the process leading to the initiation complex. First, CopG hindered binding of RNA polymerase to the promoter. Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr). A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

Show MeSH

Related in: MedlinePlus

Kinetic of dissociation of CopG from its operator at 37°C. (A) EMSA analysis of the stability of the CopG–DNA complexes. Dissociation of complexes between CopG (40 nM) and the labelled DNA was initiated by addition of a 50-fold excess of unlabelled DNA (t = 0), and samples were analyzed at the indicated times. The sum of the various CopG–DNA complexes (in brackets) was used to analyze the time course of the fraction of labelled DNA complexed to CopG. Samples of free DNA (fDNA) and of the equilibrium mixture without competitor were also loaded at t = 120. All the lanes displayed came from the same gel. (B) Time course of CopG–DNA complex dissociation. Data from three independent experiments, each performed at the indicated CopG concentration, are included. The kd values estimated were (5.5 ± 0.1) × 10−2 s−1 (16 nM CopG), (4.8 ± 0.2) × 10−2 s−1 (40 nM CopG), and (5.3 ± 0.7) × 10−2 s−1 (80 nM CopG). The solid line is the linear fit of all data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724298&req=5

Figure 4: Kinetic of dissociation of CopG from its operator at 37°C. (A) EMSA analysis of the stability of the CopG–DNA complexes. Dissociation of complexes between CopG (40 nM) and the labelled DNA was initiated by addition of a 50-fold excess of unlabelled DNA (t = 0), and samples were analyzed at the indicated times. The sum of the various CopG–DNA complexes (in brackets) was used to analyze the time course of the fraction of labelled DNA complexed to CopG. Samples of free DNA (fDNA) and of the equilibrium mixture without competitor were also loaded at t = 120. All the lanes displayed came from the same gel. (B) Time course of CopG–DNA complex dissociation. Data from three independent experiments, each performed at the indicated CopG concentration, are included. The kd values estimated were (5.5 ± 0.1) × 10−2 s−1 (16 nM CopG), (4.8 ± 0.2) × 10−2 s−1 (40 nM CopG), and (5.3 ± 0.7) × 10−2 s−1 (80 nM CopG). The solid line is the linear fit of all data.

Mentions: The kinetics of dissociation of CopG-operator complexes at 37°C could only be measured by employing a moderate (50-fold) excess of competing unlabelled DNA (Figure 4). When a higher excess (500- or 1500-fold) was used, no DNA complexed to CopG could be detected immediately on addition of the unlabelled DNA (not shown). Thus, the lifetime of CopG-operator complexes seems to depend strongly on the concentration of competing DNA, which suggests that ‘direct transfer’ of CopG to another DNA molecule contributes to the dissociation mechanism of these complexes. DNA concentration-dependent dissociation has been shown for the lac repressor and CAP proteins complexed to their target DNAs (34,35). Data for dissociation kinetic arose from three independent experiments, each performed at a different CopG concentration and thus displaying a distinct distribution of free and complexed DNA in the equilibrium mixture prior to the addition of the competing DNA. All of them gave similarly high dissociation rate constants (Figure 4). When data of all three experiments were analyzed conjointly according to Equation (1) for a pseudo first-order process, the apparent dissociation rate constant was estimated to be (5.0 ± 0.3) × 10−2 s−1 (Figure 4), which corresponded to a t1/2 ∼14 s. The low stability of the CopG-operator complexes at 37°C could account for the observed displacement of DNA-bound CopG by RNAP (Figure 3C), as the addition of the polymerase to a previously equilibrated CopG–DNA binding mixture would passively displace the equilibrium in the direction of dissociation of the CopG–DNA complexes, by sequestering free DNA.Figure 4.


Repressor CopG prevents access of RNA polymerase to promoter and actively dissociates open complexes.

Hernández-Arriaga AM, Rubio-Lepe TS, Espinosa M, del Solar G - Nucleic Acids Res. (2009)

Kinetic of dissociation of CopG from its operator at 37°C. (A) EMSA analysis of the stability of the CopG–DNA complexes. Dissociation of complexes between CopG (40 nM) and the labelled DNA was initiated by addition of a 50-fold excess of unlabelled DNA (t = 0), and samples were analyzed at the indicated times. The sum of the various CopG–DNA complexes (in brackets) was used to analyze the time course of the fraction of labelled DNA complexed to CopG. Samples of free DNA (fDNA) and of the equilibrium mixture without competitor were also loaded at t = 120. All the lanes displayed came from the same gel. (B) Time course of CopG–DNA complex dissociation. Data from three independent experiments, each performed at the indicated CopG concentration, are included. The kd values estimated were (5.5 ± 0.1) × 10−2 s−1 (16 nM CopG), (4.8 ± 0.2) × 10−2 s−1 (40 nM CopG), and (5.3 ± 0.7) × 10−2 s−1 (80 nM CopG). The solid line is the linear fit of all data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724298&req=5

Figure 4: Kinetic of dissociation of CopG from its operator at 37°C. (A) EMSA analysis of the stability of the CopG–DNA complexes. Dissociation of complexes between CopG (40 nM) and the labelled DNA was initiated by addition of a 50-fold excess of unlabelled DNA (t = 0), and samples were analyzed at the indicated times. The sum of the various CopG–DNA complexes (in brackets) was used to analyze the time course of the fraction of labelled DNA complexed to CopG. Samples of free DNA (fDNA) and of the equilibrium mixture without competitor were also loaded at t = 120. All the lanes displayed came from the same gel. (B) Time course of CopG–DNA complex dissociation. Data from three independent experiments, each performed at the indicated CopG concentration, are included. The kd values estimated were (5.5 ± 0.1) × 10−2 s−1 (16 nM CopG), (4.8 ± 0.2) × 10−2 s−1 (40 nM CopG), and (5.3 ± 0.7) × 10−2 s−1 (80 nM CopG). The solid line is the linear fit of all data.
Mentions: The kinetics of dissociation of CopG-operator complexes at 37°C could only be measured by employing a moderate (50-fold) excess of competing unlabelled DNA (Figure 4). When a higher excess (500- or 1500-fold) was used, no DNA complexed to CopG could be detected immediately on addition of the unlabelled DNA (not shown). Thus, the lifetime of CopG-operator complexes seems to depend strongly on the concentration of competing DNA, which suggests that ‘direct transfer’ of CopG to another DNA molecule contributes to the dissociation mechanism of these complexes. DNA concentration-dependent dissociation has been shown for the lac repressor and CAP proteins complexed to their target DNAs (34,35). Data for dissociation kinetic arose from three independent experiments, each performed at a different CopG concentration and thus displaying a distinct distribution of free and complexed DNA in the equilibrium mixture prior to the addition of the competing DNA. All of them gave similarly high dissociation rate constants (Figure 4). When data of all three experiments were analyzed conjointly according to Equation (1) for a pseudo first-order process, the apparent dissociation rate constant was estimated to be (5.0 ± 0.3) × 10−2 s−1 (Figure 4), which corresponded to a t1/2 ∼14 s. The low stability of the CopG-operator complexes at 37°C could account for the observed displacement of DNA-bound CopG by RNAP (Figure 3C), as the addition of the polymerase to a previously equilibrated CopG–DNA binding mixture would passively displace the equilibrium in the direction of dissociation of the CopG–DNA complexes, by sequestering free DNA.Figure 4.

Bottom Line: First, CopG hindered binding of RNA polymerase to the promoter.Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr).A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Replication of the promiscuous plasmid pMV158 requires expression of the initiator repB gene, which is controlled by the repressor CopG. Genes repB and copG are co-transcribed from promoter P(cr). We have studied the interactions between RNA polymerase, CopG and the promoter to elucidate the mechanism of repression by CopG. Complexes formed at 0 degrees C and at 37 degrees C between RNA polymerase and P(cr) differed from each other in stability and in the extent of the DNA contacted. The 37 degrees C complex was very stable (half-life of about 3 h), and shared features with typical open complexes generated at a variety of promoters. CopG protein repressed transcription from P(cr) at two different stages in the process leading to the initiation complex. First, CopG hindered binding of RNA polymerase to the promoter. Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr). A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

Show MeSH
Related in: MedlinePlus