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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.

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Kinetic of dissociation of RNAP from Pcr at 37°C. (A) EMSA analysis of the stability of the RNAP–Pcr complexes. Equilibrium mixtures contained 150 nM RNAP and 2 nM labelled DNA. Samples were analyzed at the indicated times following addition of 3 μM unlabelled DNA. Bands corresponding to free DNA (fDNA) and to specific RNAP–DNA complexes are indicated. On addition of competitor (t = 0), the complex fraction was estimated to be 0.8. In the absence of competitor, slower-migrating complexes (C*) that contained several RNAP molecules were seen. All the lanes displayed came from the same gel. (B) Time course of RNAP–Pcr complex dissociation. Linear fits of data from each of two experimental conditions are shown.
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Figure 2: Kinetic of dissociation of RNAP from Pcr at 37°C. (A) EMSA analysis of the stability of the RNAP–Pcr complexes. Equilibrium mixtures contained 150 nM RNAP and 2 nM labelled DNA. Samples were analyzed at the indicated times following addition of 3 μM unlabelled DNA. Bands corresponding to free DNA (fDNA) and to specific RNAP–DNA complexes are indicated. On addition of competitor (t = 0), the complex fraction was estimated to be 0.8. In the absence of competitor, slower-migrating complexes (C*) that contained several RNAP molecules were seen. All the lanes displayed came from the same gel. (B) Time course of RNAP–Pcr complex dissociation. Linear fits of data from each of two experimental conditions are shown.

Mentions: On the other hand, most of the binary complexes generated at 37°C resisted a brief exposition to the competitor, as ∼80% of the labelled promoter DNA remained bound to RNAP just after the addition of the unlabelled DNA (t = 0) (Figure 2A). In addition, dissociation of these RNAP–Pcr complexes was very slow (Figure 2B) and appeared to follow a first order kinetic which yielded a dissociation rate constant kd = (6.3 ± 0.7) × 10−5 s−1 (t1/2 ∼180 min). To discard this low kd being due to the stoichiometric excess of RNAP (150 nM) over the labelled promoter fragment (2 nM), experiments of dissociation of RNAP–Pcr complexes generated at 37°C were also performed by equilibrating 2 nM 32P-labelled DNA and 8 nM RNAP and, at t = 0, adding a 100-fold molar excess (200 nM) of unlabelled promoter fragment. In this case (Figure 2B), a kd = (6.0 ± 0.8) × 10−5 s−1 was estimated from the data, which was not significantly different from that obtained when 150 nM RNAP was used. Similar kd values were obtained when either 50 μg/ml or 150 μg/ml of heparin were added, instead of the unlabelled promoter fragment, to sequester free RNAP from pre-equilibrated binding mixtures containing 2 nM 32P-labelled DNA and 8 nM RNAP (data not shown). These results indicate that the apparent rate of dissociation of RNAP from Pcr is not affected by the nature or concentration of the RNAP-quenching agent, which would be thus unable to actively displace RNAP from the complexes, and would only sequester the free enzyme. Estimated kd values show that the major RNAP–Pcr complexes generated at 37°C are very stable, with a half-life of ∼3 h. The stability of these complexes is well within the range reported for the RNAP–promoter complexes generated at 37°C (6,33).Figure 2.


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 RNAP from Pcr at 37°C. (A) EMSA analysis of the stability of the RNAP–Pcr complexes. Equilibrium mixtures contained 150 nM RNAP and 2 nM labelled DNA. Samples were analyzed at the indicated times following addition of 3 μM unlabelled DNA. Bands corresponding to free DNA (fDNA) and to specific RNAP–DNA complexes are indicated. On addition of competitor (t = 0), the complex fraction was estimated to be 0.8. In the absence of competitor, slower-migrating complexes (C*) that contained several RNAP molecules were seen. All the lanes displayed came from the same gel. (B) Time course of RNAP–Pcr complex dissociation. Linear fits of data from each of two experimental conditions are shown.
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Figure 2: Kinetic of dissociation of RNAP from Pcr at 37°C. (A) EMSA analysis of the stability of the RNAP–Pcr complexes. Equilibrium mixtures contained 150 nM RNAP and 2 nM labelled DNA. Samples were analyzed at the indicated times following addition of 3 μM unlabelled DNA. Bands corresponding to free DNA (fDNA) and to specific RNAP–DNA complexes are indicated. On addition of competitor (t = 0), the complex fraction was estimated to be 0.8. In the absence of competitor, slower-migrating complexes (C*) that contained several RNAP molecules were seen. All the lanes displayed came from the same gel. (B) Time course of RNAP–Pcr complex dissociation. Linear fits of data from each of two experimental conditions are shown.
Mentions: On the other hand, most of the binary complexes generated at 37°C resisted a brief exposition to the competitor, as ∼80% of the labelled promoter DNA remained bound to RNAP just after the addition of the unlabelled DNA (t = 0) (Figure 2A). In addition, dissociation of these RNAP–Pcr complexes was very slow (Figure 2B) and appeared to follow a first order kinetic which yielded a dissociation rate constant kd = (6.3 ± 0.7) × 10−5 s−1 (t1/2 ∼180 min). To discard this low kd being due to the stoichiometric excess of RNAP (150 nM) over the labelled promoter fragment (2 nM), experiments of dissociation of RNAP–Pcr complexes generated at 37°C were also performed by equilibrating 2 nM 32P-labelled DNA and 8 nM RNAP and, at t = 0, adding a 100-fold molar excess (200 nM) of unlabelled promoter fragment. In this case (Figure 2B), a kd = (6.0 ± 0.8) × 10−5 s−1 was estimated from the data, which was not significantly different from that obtained when 150 nM RNAP was used. Similar kd values were obtained when either 50 μg/ml or 150 μg/ml of heparin were added, instead of the unlabelled promoter fragment, to sequester free RNAP from pre-equilibrated binding mixtures containing 2 nM 32P-labelled DNA and 8 nM RNAP (data not shown). These results indicate that the apparent rate of dissociation of RNAP from Pcr is not affected by the nature or concentration of the RNAP-quenching agent, which would be thus unable to actively displace RNAP from the complexes, and would only sequester the free enzyme. Estimated kd values show that the major RNAP–Pcr complexes generated at 37°C are very stable, with a half-life of ∼3 h. The stability of these complexes is well within the range reported for the RNAP–promoter complexes generated at 37°C (6,33).Figure 2.

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