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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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CopG and RNAP compete for binding to the Pcr region. (A, B) DNase I footprinting of binding mixtures containing CopG and/or RNAP. Labelled DNA was incubated at 0°C (A) or 37°C (B) in the presence (+) or in the absence (−) of CopG and RNAP prior to digestion with DNase I. When both proteins were included, CopG was added before RNAP. Cleavage products on the non-template DNA strand are shown, with regions protected by CopG or RNAP indicated by thin or thick brackets, respectively. RNAP footprints are named as in Figure 1B. Enhancements due to CopG binding are indicated by arrows. Dideoxy sequencing reactions on the same DNA are included. (C) EMSA analysis of the complexes formed at 37°C in the presence of CopG and/or RNAP. Unlabelled DNA (10 nM) was incubated with the indicated concentrations of CopG prior to addition of RNAP. Samples were treated with heparin before loading onto the gel. Bands corresponding to free DNA (fDNA), and to CopG–DNA and RNAP–DNA complexes are indicated.
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Figure 3: CopG and RNAP compete for binding to the Pcr region. (A, B) DNase I footprinting of binding mixtures containing CopG and/or RNAP. Labelled DNA was incubated at 0°C (A) or 37°C (B) in the presence (+) or in the absence (−) of CopG and RNAP prior to digestion with DNase I. When both proteins were included, CopG was added before RNAP. Cleavage products on the non-template DNA strand are shown, with regions protected by CopG or RNAP indicated by thin or thick brackets, respectively. RNAP footprints are named as in Figure 1B. Enhancements due to CopG binding are indicated by arrows. Dideoxy sequencing reactions on the same DNA are included. (C) EMSA analysis of the complexes formed at 37°C in the presence of CopG and/or RNAP. Unlabelled DNA (10 nM) was incubated with the indicated concentrations of CopG prior to addition of RNAP. Samples were treated with heparin before loading onto the gel. Bands corresponding to free DNA (fDNA), and to CopG–DNA and RNAP–DNA complexes are indicated.

Mentions: To disclose the mechanism of CopG-mediated repression of Pcr we first investigated the step at which the repressor prevents transcription from this promoter. To this end, the promoter fragment (2 nM) was equilibrated, at 0°C or 37°C, with excess CopG (120 nM), so that virtual saturation of the operators was obtained. Subsequently, RNAP was added, also at a molar excess (150 nM) relative to the promoter DNA, and incubation continued for 60 more minutes at 0°C or for 30 more minutes at 37°C, prior to DNase I probing of the protein footprints (Figure 3A and B). At either temperature, the footprint pattern of the sample containing both proteins was almost identical to that of the DNA incubated with CopG alone. By decreasing the repressor concentration, the RNAP-specific footprint pattern was increasingly apparent (not shown). These results indicated that CopG was able to prevent subsequent binding of RNAP to Pcr. To verify that addition of RNAP to a preformed CopG–DNA complex does not result in the formation of a ternary repressor–DNA–RNAP complex, we analyzed the electrophoretic mobility of DNA samples that were incubated in the absence of CopG or in the presence of increasing concentrations of the repressor prior to the addition of RNAP (Figure 3C). The samples were treated with heparin before loading onto the gel, so that unspecific complexes resulting from the binding of additional molecules of RNAP to non-promoter regions were removed and could not interfere with identification of any existing ternary complex generated by simultaneous binding of both proteins to the operator/promoter region of the DNA. As heparin treatment also removes unstable specific closed complexes, electrophoretic mobility shift assay (EMSA) was only performed with binding mixtures prepared at 37°C. Bands that migrate as the specific binary CopG–DNA or RNAP–DNA complexes, but no super-shifted bands indicative of stable ternary complexes, were seen at all CopG concentrations. Increasing concentrations of the repressor resulted in a decrease of the fraction of DNA bound to RNAP and a concomitant increase of the fraction of CopG–DNA complexes (Figure 3C). Similarly, in experiments in which increasing concentrations of RNAP were added to a CopG–DNA equilibrium mixture, the fraction of RNAP–DNA complex was seen to increase while that of the CopG–DNA complexes decreased (not shown). Thus, the success of CopG in preventing formation of a stable RNAP–Pcr complex depends on its own concentration and that of RNAP. These results, together with those of the DNase I footprinting assays performed at 0°C or 37°C in the presence of both proteins, show that binding of CopG and RNAP are mutually exclusive, and that CopG competes with RNAP for binding to the target DNA. RNAP was able to displace DNA-bound CopG, as revealed by the appearance of RNAP–DNA complexes upon addition of the enzyme to binding mixtures in which virtually all the operator sites were saturated with CopG (Figure 3C, compare the samples containing 80 nM CopG, with and without RNAP). A passive displacement of the repressor from its complexes with the operator, resulting from the binding of RNAP to the free DNA present in the CopG equilibrium binding mixture, could account for this observation, provided that CopG–DNA complexes dissociate rapidly on the time scale of the incubation with RNAP.Figure 3.


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)

CopG and RNAP compete for binding to the Pcr region. (A, B) DNase I footprinting of binding mixtures containing CopG and/or RNAP. Labelled DNA was incubated at 0°C (A) or 37°C (B) in the presence (+) or in the absence (−) of CopG and RNAP prior to digestion with DNase I. When both proteins were included, CopG was added before RNAP. Cleavage products on the non-template DNA strand are shown, with regions protected by CopG or RNAP indicated by thin or thick brackets, respectively. RNAP footprints are named as in Figure 1B. Enhancements due to CopG binding are indicated by arrows. Dideoxy sequencing reactions on the same DNA are included. (C) EMSA analysis of the complexes formed at 37°C in the presence of CopG and/or RNAP. Unlabelled DNA (10 nM) was incubated with the indicated concentrations of CopG prior to addition of RNAP. Samples were treated with heparin before loading onto the gel. Bands corresponding to free DNA (fDNA), and to CopG–DNA and RNAP–DNA complexes are indicated.
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Figure 3: CopG and RNAP compete for binding to the Pcr region. (A, B) DNase I footprinting of binding mixtures containing CopG and/or RNAP. Labelled DNA was incubated at 0°C (A) or 37°C (B) in the presence (+) or in the absence (−) of CopG and RNAP prior to digestion with DNase I. When both proteins were included, CopG was added before RNAP. Cleavage products on the non-template DNA strand are shown, with regions protected by CopG or RNAP indicated by thin or thick brackets, respectively. RNAP footprints are named as in Figure 1B. Enhancements due to CopG binding are indicated by arrows. Dideoxy sequencing reactions on the same DNA are included. (C) EMSA analysis of the complexes formed at 37°C in the presence of CopG and/or RNAP. Unlabelled DNA (10 nM) was incubated with the indicated concentrations of CopG prior to addition of RNAP. Samples were treated with heparin before loading onto the gel. Bands corresponding to free DNA (fDNA), and to CopG–DNA and RNAP–DNA complexes are indicated.
Mentions: To disclose the mechanism of CopG-mediated repression of Pcr we first investigated the step at which the repressor prevents transcription from this promoter. To this end, the promoter fragment (2 nM) was equilibrated, at 0°C or 37°C, with excess CopG (120 nM), so that virtual saturation of the operators was obtained. Subsequently, RNAP was added, also at a molar excess (150 nM) relative to the promoter DNA, and incubation continued for 60 more minutes at 0°C or for 30 more minutes at 37°C, prior to DNase I probing of the protein footprints (Figure 3A and B). At either temperature, the footprint pattern of the sample containing both proteins was almost identical to that of the DNA incubated with CopG alone. By decreasing the repressor concentration, the RNAP-specific footprint pattern was increasingly apparent (not shown). These results indicated that CopG was able to prevent subsequent binding of RNAP to Pcr. To verify that addition of RNAP to a preformed CopG–DNA complex does not result in the formation of a ternary repressor–DNA–RNAP complex, we analyzed the electrophoretic mobility of DNA samples that were incubated in the absence of CopG or in the presence of increasing concentrations of the repressor prior to the addition of RNAP (Figure 3C). The samples were treated with heparin before loading onto the gel, so that unspecific complexes resulting from the binding of additional molecules of RNAP to non-promoter regions were removed and could not interfere with identification of any existing ternary complex generated by simultaneous binding of both proteins to the operator/promoter region of the DNA. As heparin treatment also removes unstable specific closed complexes, electrophoretic mobility shift assay (EMSA) was only performed with binding mixtures prepared at 37°C. Bands that migrate as the specific binary CopG–DNA or RNAP–DNA complexes, but no super-shifted bands indicative of stable ternary complexes, were seen at all CopG concentrations. Increasing concentrations of the repressor resulted in a decrease of the fraction of DNA bound to RNAP and a concomitant increase of the fraction of CopG–DNA complexes (Figure 3C). Similarly, in experiments in which increasing concentrations of RNAP were added to a CopG–DNA equilibrium mixture, the fraction of RNAP–DNA complex was seen to increase while that of the CopG–DNA complexes decreased (not shown). Thus, the success of CopG in preventing formation of a stable RNAP–Pcr complex depends on its own concentration and that of RNAP. These results, together with those of the DNase I footprinting assays performed at 0°C or 37°C in the presence of both proteins, show that binding of CopG and RNAP are mutually exclusive, and that CopG competes with RNAP for binding to the target DNA. RNAP was able to displace DNA-bound CopG, as revealed by the appearance of RNAP–DNA complexes upon addition of the enzyme to binding mixtures in which virtually all the operator sites were saturated with CopG (Figure 3C, compare the samples containing 80 nM CopG, with and without RNAP). A passive displacement of the repressor from its complexes with the operator, resulting from the binding of RNAP to the free DNA present in the CopG equilibrium binding mixture, could account for this observation, provided that CopG–DNA complexes dissociate rapidly on the time scale of the incubation with RNAP.Figure 3.

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