Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer

Steinmetzer, Katrin; Behlke, Joachim; Brantl, Sabine

doi:10.1006/jmbi.1998.2122

Cited by 22 publications

(49 citation statements)

References 31 publications

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“…These values are typical of prokaryotic repressors with helixturn-helix recognition motifs (25,27). Comparison of the global dissociation constants indicates that BlaI presents increasing affinities for OP3 Ͻ OP2 Ͻ OP1.…”

Section: Regulation Of ␤-Lactamase In Gram-positive Bacteriamentioning

confidence: 87%

“…The former is often cooperative and is observed for the LexA bacterial transcriptional repressor (23,24). Many regulators bind DNA according to the dimer pathway (25). Because a helix-turn-helix recognition motif interacts only with five or six base pairs, the ability of this class of DNA-binding proteins to form dimers and higher order oligomers is fundamental to stabilize its binding to target DNA.…”

Section: Discussionmentioning

confidence: 99%

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Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

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Vreuls

Herman

et al. 2003

Journal of Biological Chemistry

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In the absence of penicillin, the ␤-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 M by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the ␤-lactamase locus (blaP-blaIblaR1) were lower than (1.9 M) or in the same range as (75 M) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K dOP1 ‫؍‬ 1.7 ؎ 0.5 10 The blaP gene encodes the class A ␤-lactamase of Bacillus licheniformis 749/I. In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1-3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4, 5). The blaP, blaI, and blaR1 genes are clustered in a divergeon (bla divergeon) in which blaI and blaR1 form an operon (Fig. 1). The blaR1 gene encodes a transmembrane protein that acts as penicillin receptor (6 -8), and blaR2 is not identified yet and is not linked to the bla divergeon. The BlaR2 protein is essential for the inactivation of BlaI. In Staphylococcus aureus, the blaZ and mecA genes, encoding respectively a ␤-lactamase and the low affinity penicillinbinding protein 2Ј, are regulated by similar elements (9, 10).The blaI gene encodes a 128-residue protein characterized by two functional and separate domains (11). The DNA-binding domain, a helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the Cterminal region. After deletion of the C-terminal domain, the repressor becomes unable to dimerize and to form a stable complex with its operators. DNase footprinting experiments and filter binding reactions revealed the presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and show the deduced 5Ј-AAAGTATTACATATGTAACNTTT-3Ј consensus sequence (Fig. 1).In the presence of ␤-lactam antibiotics, the C-terminal domain of BlaR1 is acylated (6, 7). This event triggers the activation of the putative cytoplasmic metalloprotease motif of BlaR1 by self-proteolysis (8, 12). In S. aureus, the ␤-lactamase induction correlates with BlaI proteolysis between residues Asn 101 and Phe 102 , and it is postulated that the activated form of BlaR1 proteolyses BlaI (12-...

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Section: Regulation Of ␤-Lactamase In Gram-positive Bacteriamentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Filée

Vreuls

Herman

et al. 2003

Journal of Biological Chemistry

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“…Overexpression and purification of the proteins from E. coli were performed as described before (Steinmetzer et al, 1998). Protein concentrations were determined by Bradford assays based on calibration curves obtained with the values from acidic hydrolysis of His ' -CopR.…”

Section: Construction Of E Coli Vectors For Overexpression Of Mutatedmentioning

confidence: 99%

“…CopR represses transcription of the essential repR mRNA (Brantl, 1994) and prevents convergent transcription of sense and antisense RNAs (Brantl & Wagner, 1997). Previously, we showed that CopR binds asymmetrically as a preformed dimer at two consecutive major grooves of the DNA (Steinmetzer & Brantl, 1997 ;Steinmetzer et al, 1998) and identified amino acids involved in DNA binding and dimerization (Steinmetzer et al, 2000a, b). The last 29 aa of CopR are essential for neither DNA binding nor dimerization, but are essential for protein stability (Kuhn et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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

2001

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Replication of the streptococcal plasmid pIP501 is regulated by two components, CopR and the antisense RNA, RNAIII. CopR represses transcription of the essential repR mRNA about 10-to 20-fold and, additionally, prevents convergent transcription of sense and antisense RNAs. It has been demonstrated that CopR binds as a preformed dimer. DNA binding and dimerization constants were determined and amino acids were identified that are involved in DNA binding and dimerization. It was demonstrated that the Cterminal 20 aa of CopR are not involved in either activity, but play an important role for CopR stability. Furthermore, it was found that the C terminus of CopR is structured containing a β-strand structure, most probably between the alternating hydrophilic and hydrophobic amino acids 76 and 84 (QVTLELEME). In this study stability motifs within the C terminus of CopR were dissected. Both the cognate and a heterologous (QVTVTVTVT) β-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted α-helix (QVTLKLKMK) or a predicted unstructured sequence (QVTPEPEPE) caused severe and moderate destabilization, respectively. E80 seemed to be the only important C-terminal glutamic acid residue. Deletion of seven C-terminal amino acids from either wild-type CopR or CopVT reduced the half-life to " 50 % indicating that this C-terminal sequence is a second stability motif.

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“…The operator sequence was narrowed down to 17 bp. Furthermore, it was found that CopR binds exclusively as a dimer, and the equilibrium dissociation constants for the CopR dimers and the CopR-DNA complex were calculated to be 0.4 nM and 1.4 M, respectively (29). A three-dimensional model of the Nterminal 63 amino acids of CopR was built and was used to identify amino acids involved in DNA binding and dimerization (30,31,32).…”

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confidence: 99%

Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

Freede

Brantl

2004

J Bacteriol

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CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10-to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 M, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3 positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.Replication of the streptococcal plasmid pIP501 is regulated by two components that act in concert: the transcriptional repressor CopR (10.6 kDa) and the antisense RNA RNAIII (136 nucleotides [nt]) (5). Whereas RNAIII exerts its inhibitory effect by premature termination (attenuation) of the essential repR mRNA (6, 8), CopR has a dual function. On the one hand, it represses transcription from the essential repR promoter pII about 10-to 20-fold (7); on the other hand, it prevents convergent transcription from pII and pIII (antisense promoter), thereby indirectly increasing transcription initiation at pIII (9). Previously, it was found that CopR contacts the DNA asymmetrically at two consecutive major grooves that share the consensus motif 5Ј-CGTG (28). Thus, the outermost G residues were found to be most important for CopR binding, whereas exchanges of nucleotides adjacent (3Ј) to the CGTG motif only slightly altered DNA binding. The operator sequence was narrowed down to 17 bp. Furthermore, it was found that CopR binds exclusively as a dimer, and the equilibrium dissociation constants for the CopR dimers and the CopR-DNA complex were calculated to be 0.4 nM and 1.4 M, respectively (29). A three-dimensional model of the Nterminal 63 amino acids of CopR was built and was used to identify amino acids involved in DNA binding and dimerization (30,31,32). By this means, it was found that amino acids R29 and R34, located in the recognition helix (helix III) of the helix-turn-helix motif, make specific contacts to the DNA at G240 (binding site I) and G254 (binding site II) or G242/T243 (site I) and G251 (site II), respectively. Water-mediated contacts were suggested for E35 interacting with the outermost C residues in both binding sites. ...

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Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer

Cited by 22 publications

References 31 publications

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

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

Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

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