Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

Kuhn, Kornelia; Steinmetzer, Katrin; Brantl, Sabine

doi:10.1099/00221287-147-12-3387

Cited by 7 publications

(6 citation statements)

References 24 publications

(27 reference statements)

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“…Their amino acid sequences diverge only at the C terminus, which is solely responsible for protein stability (Kuhn et al, 2000(Kuhn et al, , 2001. Furthermore, the three Cop operator sites are almost identical, differing only in the spacer region between binding sites I and II and in the flanking region.…”

Section: Discussionmentioning

confidence: 99%

“…A 3D model of the N-terminal 63 amino acids of CopR was built, allowing the identification of amino acids involved in DNA binding and dimerization (Steinmetzer et al, 2000a(Steinmetzer et al, , b, 2002a. Furthermore, it was established that the structured acidic C terminus of CopR that forms a b-strand is necessary for stabilization of the protein (Kuhn et al, 2000(Kuhn et al, , 2001. A fluorescence energy study revealed that CopR bends the operator DNA slightly -20-25 u -upon binding (Steinmetzer et al, 2002b).…”

Section: Introductionmentioning

confidence: 99%

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Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

2011

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CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10-to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR-operator complexes are 18-fold less stable than RNA polymerase (RNAP)-pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO 4 footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

2011

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“…CD measurements revealed that the C-terminus of CopR that contains alternating hydrophilic and hydrophobic aa residues is structured and forms a β-strand (Kuhn et al, 2000 ). Further analysis of the stabilizing motifs within the C-terminus (Kuhn et al, 2001 ) showed that both the wild-type (QVTLELEME, Figure 3A ) and an artificial (QVTVTVTVT) β-strand structure (variant CopRVT) between aa 76 and 84 stabilized the corresponding protein derivatives. By contrast, replacement of the β-strand by an α-helix or an unstructured sequence significantly or moderately destabilized the protein.…”

Section: Pip501 Replication and Copy Number Controlmentioning

confidence: 99%

DNA-Binding Proteins Regulating pIP501 Transfer and Replication

Grohmann

Goessweiner‐Mohr²,

Brantl

2016

Front. Mol. Biosci.

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pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently detected in clinical Enterococcus faecalis and Enterococcus faecium strains. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual function: It acts as transcriptional repressor at the repR promoter and, in addition, prevents convergent transcription of RNAIII and repR mRNA (RNAII), which indirectly increases RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including Streptomyces and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter overlapping with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein) and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.

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“…Eight amino acids are involved in forming the dimeric interface, and two of them required for correct folding of the monomer (41,42). The structured acidic C terminus of CopR is important for protein stability and contains a stretch of alternating hydrophilic and hydrophobic amino acids that form a β-strand (43,44). Its deletion does not impair the in vivo function of CopR, but decreases CopR half-life from 42 min to 4 to 5 min (43).…”

Section: Introductionmentioning

confidence: 99%

Plasmid Replication Control by Antisense RNAs

Brantl

2014

Microbiol Spectr

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Plasmids are selfish genetic elements that normally constitute a burden for the bacterial host cell. This burden is expected to favor plasmid loss. Therefore, plasmids have evolved mechanisms to control their replication and ensure their stable maintenance. Replication control can be either mediated by iterons or by antisense RNAs. Antisense RNAs work through a negative control circuit. They are constitutively synthesized and metabolically unstable. They act both as a measuring device and a regulator, and regulation occurs by inhibition. Increased plasmid copy numbers lead to increasing antisense-RNA concentrations, which, in turn, result in the inhibition of a function essential for replication. On the other hand, decreased plasmid copy numbers entail decreasing concentrations of the inhibiting antisense RNA, thereby increasing the replication frequency. Inhibition is achieved by a variety of mechanisms, which are discussed in detail. The most trivial case is the inhibition of translation of an essential replication initiator protein (Rep) by blockage of the rep-ribosome binding site. Alternatively, ribosome binding to a leader peptide mRNA whose translation is required for efficient Rep translation can be prevented by antisense-RNA binding. In 2004, translational attenuation was discovered. Antisense-RNA-mediated transcriptional attenuation is another mechanism that has, so far, only been detected in plasmids of Gram-positive bacteria. ColE1, a plasmid that does not need a plasmid-encoded replication initiator protein, uses the inhibition of primer formation. In other cases, antisense RNAs inhibit the formation of an activator pseudoknot that is required for efficient Rep translation.

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Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

Cited by 7 publications

References 24 publications

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

DNA-Binding Proteins Regulating pIP501 Transfer and Replication

Plasmid Replication Control by Antisense RNAs

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