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Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

Riley SP, Bykowski T, Cooley AE, Burns LH, Babb K, Brissette CA, Bowman A, Rotondi M, Miller MC, DeMoll E, Lim K, Fried MG, Stevenson B - Nucleic Acids Res. (2009)

Bottom Line: Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA.These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein.EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.

ABSTRACT
The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

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Sequences of DNA probes used for defining the sequence requirements for high-affinity binding by EbfC. All were based upon the sequence of the B. burgdorferi strain B31 erpAB Operator 2. The ability of each DNA to form high-affinity EbfC–DNA interactions is indicated. All probes were labeled with a biotin moiety at the right end. The uppermost sequence is the complete sequence of the wild-type probe b-WT. Other than the b-SRK series, all other EMSA probes were identical to b-WT except where indicated by underlining and lower case lettering. Sequences of EbfC-binding sites I, II and III are shown in bold black type. Parts of the pCR2.1-derived sequences of the b-SRK series are indicated by lower case italics.
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Figure 1: Sequences of DNA probes used for defining the sequence requirements for high-affinity binding by EbfC. All were based upon the sequence of the B. burgdorferi strain B31 erpAB Operator 2. The ability of each DNA to form high-affinity EbfC–DNA interactions is indicated. All probes were labeled with a biotin moiety at the right end. The uppermost sequence is the complete sequence of the wild-type probe b-WT. Other than the b-SRK series, all other EMSA probes were identical to b-WT except where indicated by underlining and lower case lettering. Sequences of EbfC-binding sites I, II and III are shown in bold black type. Parts of the pCR2.1-derived sequences of the b-SRK series are indicated by lower case italics.

Mentions: Sequences of biotinylated DNAs used to define optimal EbfC-binding activities by electrophoretic mobility shift assays (EMSA) are illustrated in Figure 1. DNAs were produced by PCR amplification using appropriate primers, templates and LA Taq polymerase (Takara). Amplification consisted of 40 cycles of 94°C for 1 min, 55°C for 1 min and 68°C for 1 min. Reaction products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. DNAs were extracted into nuclease-free water using Wizard SV gel purification system (Promega). DNA concentrations were determined spectrophotometrically by measuring absorbance at 260 nm, with an absorbance of 1.0 = 50 μg/ml DNA, then each diluted to a final concentration of 1 nM (10× stock).Figure 1.


Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

Riley SP, Bykowski T, Cooley AE, Burns LH, Babb K, Brissette CA, Bowman A, Rotondi M, Miller MC, DeMoll E, Lim K, Fried MG, Stevenson B - Nucleic Acids Res. (2009)

Sequences of DNA probes used for defining the sequence requirements for high-affinity binding by EbfC. All were based upon the sequence of the B. burgdorferi strain B31 erpAB Operator 2. The ability of each DNA to form high-affinity EbfC–DNA interactions is indicated. All probes were labeled with a biotin moiety at the right end. The uppermost sequence is the complete sequence of the wild-type probe b-WT. Other than the b-SRK series, all other EMSA probes were identical to b-WT except where indicated by underlining and lower case lettering. Sequences of EbfC-binding sites I, II and III are shown in bold black type. Parts of the pCR2.1-derived sequences of the b-SRK series are indicated by lower case italics.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2665219&req=5

Figure 1: Sequences of DNA probes used for defining the sequence requirements for high-affinity binding by EbfC. All were based upon the sequence of the B. burgdorferi strain B31 erpAB Operator 2. The ability of each DNA to form high-affinity EbfC–DNA interactions is indicated. All probes were labeled with a biotin moiety at the right end. The uppermost sequence is the complete sequence of the wild-type probe b-WT. Other than the b-SRK series, all other EMSA probes were identical to b-WT except where indicated by underlining and lower case lettering. Sequences of EbfC-binding sites I, II and III are shown in bold black type. Parts of the pCR2.1-derived sequences of the b-SRK series are indicated by lower case italics.
Mentions: Sequences of biotinylated DNAs used to define optimal EbfC-binding activities by electrophoretic mobility shift assays (EMSA) are illustrated in Figure 1. DNAs were produced by PCR amplification using appropriate primers, templates and LA Taq polymerase (Takara). Amplification consisted of 40 cycles of 94°C for 1 min, 55°C for 1 min and 68°C for 1 min. Reaction products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. DNAs were extracted into nuclease-free water using Wizard SV gel purification system (Promega). DNA concentrations were determined spectrophotometrically by measuring absorbance at 260 nm, with an absorbance of 1.0 = 50 μg/ml DNA, then each diluted to a final concentration of 1 nM (10× stock).Figure 1.

Bottom Line: Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA.These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein.EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.

ABSTRACT
The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

Show MeSH
Related in: MedlinePlus