Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA 1 1Edited by T. Richmond

Steinmetzer, Katrin; Brantl, Sabine

doi:10.1006/jmbi.1997.1083

Cited by 24 publications

(35 citation statements)

References 24 publications

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“…Such exchanges were not found with SELEX II or III. The importance of the G's in positions 240 and 254 had been demonstrated before (28). G-to-A exchanges at these two positions abolished binding or decreased the binding affinity drastically.…”

Section: Resultsmentioning

confidence: 70%

“…Such a perfectly symmetric operator sequence with T243C (Fig. 2), termed KS9, had been analyzed previously (28) and was found to be bound at least as efficiently as the wild-type sequence. Our three-dimensional model of the N-terminal 63 amino acids of CopR (30) predicts that this nucleotide position in binding site I is contacted by R34 of the recognition helix (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Plasmid pQEC1 was used for overexpression and purification of His 6 CopR as described previously (28,29). For the preparation of native CopR, strain ER2566 (pTYBC11) was used.…”

Section: Methodsmentioning

confidence: 99%

“…As a reference for the excision of the shifted CopR target, which was not visible in the first round of selection with SELEX II and III, primers with wild-type operator sequence but that were the same length as the SELEX primer were used in each SELEX experiment and treated in the same way (Klenow reaction, PCR amplification, labeling, and EMSA). After round 3 of each SELEX procedure, a 0.6 M wild-type DNA fragment (KS1) (28), which has no binding sites for amplification primers, was added as a competitor in each binding reaction to promote selection towards a high-affinity and high-specificity pool. After 30 min at 30°C, bound and unbound DNA species were separated on 8% native polyacrylamide gels at 230 V. Band shifts were detected by phosphorimaging.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 98%

“…Plasmid pQEC1 was used for overexpression and purification of His 6 CopR as described previously (28,29). For the preparation of native CopR, strain ER2566 (pTYBC11) was used.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Steinmetzer

Hillisch²,

Behlke

et al. 2000

Proteins

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The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.

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Transcriptional repressor CopR: Amino acids involved in forming the dimeric interface

Steinmetzer

Hillisch²,

Behlke

et al. 2000

Proteins

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Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. It acts as a transcriptional repressor at the essential repR promoter. Furthermore, CopR prevents convergent transcription from the repR and the antisense promoter, thereby indirectly increasing the amount of antisense-RNA, the second regulatory component. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'CGTG. Previously, a CopR structural model was built and used to identify amino acids involved in DNA binding. These data showed that CopR is a HTH protein belonging to the lambda repressor superfamily and allowed the identification of two amino acids involved in specific DNA recognition. Here, we describe site-directed mutagenesis in combination with EMSA, dimerization studies using sedimentation equilibrium, and CD measurements to verify the model predictions concerning amino acids involved in dimerization. With this approach, the dimeric interface could be located between amino acids I44 and L62. F5 located at the N-terminus is additionally required for proper folding, and could, therefore, not be unequivocally assigned to the dimeric interface. CD measurements at protein concentrations well below K(Dimer) revealed that the monomer of CopR is folded.

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Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA 1 1Edited by T. Richmond

Cited by 24 publications

References 24 publications

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

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

Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Transcriptional repressor CopR: Amino acids involved in forming the dimeric interface

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