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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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The Pcr promoter of pMV158. (A) Conserved elements of Pcr. The copG start codon, the +1 site, and the −35 and extended −10 elements are in capital letters; distances between them are shown. The SE of the CopG operator is boxed. (B) Footprinting pattern of the 0°C and 37°C RNAP–Pcr complexes. The scheme summarizes the results shown in Supplementary Figures S1 and S2. Promoter positions are numbered relative to the transcription start site. DNase I protections are denoted by numbered brackets. Thin dotted-line brackets indicate weak protections. Enhancements to HO• attack are shown by arrows.
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Figure 1: The Pcr promoter of pMV158. (A) Conserved elements of Pcr. The copG start codon, the +1 site, and the −35 and extended −10 elements are in capital letters; distances between them are shown. The SE of the CopG operator is boxed. (B) Footprinting pattern of the 0°C and 37°C RNAP–Pcr complexes. The scheme summarizes the results shown in Supplementary Figures S1 and S2. Promoter positions are numbered relative to the transcription start site. DNase I protections are denoted by numbered brackets. Thin dotted-line brackets indicate weak protections. Enhancements to HO• attack are shown by arrows.

Mentions: Transcriptional repressor CopG, encoded by the promiscuous plasmid pMV158, is a small protein (45 amino-acid polypeptide chains) which, by inhibiting synthesis of the cop-rep mRNA, regulates both the expression of its own gene and that of the initiator of replication (repB) gene. Contacts of CopG to its target DNA span about 50 bp, through a region that includes the –35 and –10 boxes of the regulated Pcr promoter (15) (Figure 1A). In the centre of the contacted DNA, and overlapping the –35 box, there is a 13-bp pseudo-symmetric element (SE; Figure 1A) which constitutes the primary target of CopG. Although the structure solved from the co-crystals of CopG bound to either a 19- or a 22-bp double-stranded DNA shows two protein dimers, each interacting with a half of the SE, the working model for the whole DNA specifically contacted by CopG assumes the cooperative binding of four dimers of the protein (16,17). CopG belongs to the ribbon-helix-helix (RHH) class of DNA-binding proteins, which interact specifically with the bases of the DNA through residues located in the two-stranded antiparallel β-sheet (18,19). Members of the RHH class exhibit cooperativity based on protein–protein interactions, generating at least a dimer of protein dimers upon binding to their target DNA (20–22). CopG, which is no more than the RHH motif, is the smallest among these proteins and represents the minimal DNA-binding structure within this class of proteins (17). The peptide-backbone structure of the CopG dimer is almost identical to that of the RHH motif of Arc repressor from Salmonella bacteriophage P22, although this latter protein is slightly bigger (53 residues polypeptide chains) and contains an additional N-terminal region which also interacts with the target DNA (17,19). The similarity between the Arc- and CopG-mediated transcriptional regulatory systems also extends to the DNA moiety. In both systems, the operator to which the repressor binds overlaps the promoter region and contains two inversely-repeated copies of a 4-bp sequence (5′-TAGA-3′ for Arc operator, and the self-palindrome 5′-TGCA-3′ for CopG operator) which are 5 bp apart. This arrangement results in 13-bp SEs, whose dyad axes pass in both operators through a central G:C base pair. A dimer of either Arc or CopG interacts with each half of the respective SE establishing direct contacts with at least one base of each of the base pairs constituting the 4-bp inversely-repeated boxes, and with two further bases outside the 13-bp palindromic element (one at each side of the SE) (16,19). With respect to the mechanism of transcription inhibition, some detailed information has been published for the Arc-regulated system. Binding of Arc to its operator represses transcription from two divergent and overlapping promoters, Pant and Pmnt (23). It has been shown that binding of Arc does not preclude RNAP binding to Pant, but prevents the polymerase from forming a heparin-resistant stable complex at this promoter, so that the rate of generation of a transcriptionally-competent open complex is reduced (23). Although CopG-mediated repression of transcription from Pcr has been reported both in vivo and in vitro (15), the mechanisms of this repression have not been investigated previously. Here we characterize the complexes formed by binding of RNAP to the Pcr promoter at 0°C and 37°C. In addition, we present results showing that CopG does not only prevent binding of RNAP to the promoter, but is also able to dislodge the polymerase once it has formed a stable heparin-resistant complex.Figure 1.


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)

The Pcr promoter of pMV158. (A) Conserved elements of Pcr. The copG start codon, the +1 site, and the −35 and extended −10 elements are in capital letters; distances between them are shown. The SE of the CopG operator is boxed. (B) Footprinting pattern of the 0°C and 37°C RNAP–Pcr complexes. The scheme summarizes the results shown in Supplementary Figures S1 and S2. Promoter positions are numbered relative to the transcription start site. DNase I protections are denoted by numbered brackets. Thin dotted-line brackets indicate weak protections. Enhancements to HO• attack are shown by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: The Pcr promoter of pMV158. (A) Conserved elements of Pcr. The copG start codon, the +1 site, and the −35 and extended −10 elements are in capital letters; distances between them are shown. The SE of the CopG operator is boxed. (B) Footprinting pattern of the 0°C and 37°C RNAP–Pcr complexes. The scheme summarizes the results shown in Supplementary Figures S1 and S2. Promoter positions are numbered relative to the transcription start site. DNase I protections are denoted by numbered brackets. Thin dotted-line brackets indicate weak protections. Enhancements to HO• attack are shown by arrows.
Mentions: Transcriptional repressor CopG, encoded by the promiscuous plasmid pMV158, is a small protein (45 amino-acid polypeptide chains) which, by inhibiting synthesis of the cop-rep mRNA, regulates both the expression of its own gene and that of the initiator of replication (repB) gene. Contacts of CopG to its target DNA span about 50 bp, through a region that includes the –35 and –10 boxes of the regulated Pcr promoter (15) (Figure 1A). In the centre of the contacted DNA, and overlapping the –35 box, there is a 13-bp pseudo-symmetric element (SE; Figure 1A) which constitutes the primary target of CopG. Although the structure solved from the co-crystals of CopG bound to either a 19- or a 22-bp double-stranded DNA shows two protein dimers, each interacting with a half of the SE, the working model for the whole DNA specifically contacted by CopG assumes the cooperative binding of four dimers of the protein (16,17). CopG belongs to the ribbon-helix-helix (RHH) class of DNA-binding proteins, which interact specifically with the bases of the DNA through residues located in the two-stranded antiparallel β-sheet (18,19). Members of the RHH class exhibit cooperativity based on protein–protein interactions, generating at least a dimer of protein dimers upon binding to their target DNA (20–22). CopG, which is no more than the RHH motif, is the smallest among these proteins and represents the minimal DNA-binding structure within this class of proteins (17). The peptide-backbone structure of the CopG dimer is almost identical to that of the RHH motif of Arc repressor from Salmonella bacteriophage P22, although this latter protein is slightly bigger (53 residues polypeptide chains) and contains an additional N-terminal region which also interacts with the target DNA (17,19). The similarity between the Arc- and CopG-mediated transcriptional regulatory systems also extends to the DNA moiety. In both systems, the operator to which the repressor binds overlaps the promoter region and contains two inversely-repeated copies of a 4-bp sequence (5′-TAGA-3′ for Arc operator, and the self-palindrome 5′-TGCA-3′ for CopG operator) which are 5 bp apart. This arrangement results in 13-bp SEs, whose dyad axes pass in both operators through a central G:C base pair. A dimer of either Arc or CopG interacts with each half of the respective SE establishing direct contacts with at least one base of each of the base pairs constituting the 4-bp inversely-repeated boxes, and with two further bases outside the 13-bp palindromic element (one at each side of the SE) (16,19). With respect to the mechanism of transcription inhibition, some detailed information has been published for the Arc-regulated system. Binding of Arc to its operator represses transcription from two divergent and overlapping promoters, Pant and Pmnt (23). It has been shown that binding of Arc does not preclude RNAP binding to Pant, but prevents the polymerase from forming a heparin-resistant stable complex at this promoter, so that the rate of generation of a transcriptionally-competent open complex is reduced (23). Although CopG-mediated repression of transcription from Pcr has been reported both in vivo and in vitro (15), the mechanisms of this repression have not been investigated previously. Here we characterize the complexes formed by binding of RNAP to the Pcr promoter at 0°C and 37°C. In addition, we present results showing that CopG does not only prevent binding of RNAP to the promoter, but is also able to dislodge the polymerase once it has formed a stable heparin-resistant complex.Figure 1.

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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