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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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Related in: MedlinePlus

Competition of EbfC binding to probe b-WT by the non-specific competitor poly-dI-dC. EMSA included 100 pM b-WT DNA, and lanes 2–10 contained 1 μM EbfC; also contained either 0, 1, 5, 10, 100, 500, 1000, 5000 or 25 000 pg/μl poly-dI-dC, respectively.
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Figure 3: Competition of EbfC binding to probe b-WT by the non-specific competitor poly-dI-dC. EMSA included 100 pM b-WT DNA, and lanes 2–10 contained 1 μM EbfC; also contained either 0, 1, 5, 10, 100, 500, 1000, 5000 or 25 000 pg/μl poly-dI-dC, respectively.

Mentions: Initially, mutants of PerpAB were produced in which either site I, site II or both sites I and II were changed to 5′-CACAACA-3′, all in the context of otherwise normal PerpAB DNA sequences (Figure 1). EMSA using a probe with the wild-type PerpAB sequence, b-WT, yielded two observable DNA–EbfC complexes, with the slower mobility (upper) complex having a higher affinity interaction (Figure 2A). Probes that contain only one TGTA/TACA palindrome (b-C1 and b-C2) exhibited the same two EbfC–DNA complexes, but at decreased relative levels (Figure 3A). The signal strengths of the slower mobility complexes for EbfC binding to either probes b-C1 or b-C2 were approximately one half the value for b-WT. There were no apparent differences in EbfC binding to DNA probes that contained only wild type site I or site II, indicating relatively equal affinities for both sites. However, the mutation of both TGTA/TACA palindromes in probe b-C1/2 dramatically reduced EbfC binding (Figure 2A). These results confirm that EbfC preferentially binds the previously identified palindromic sequence and demonstrate that the apparent binding affinity when two sites are present is greater than when DNA contains only one site. This was likely due to the higher relative concentration of high-affinity binding sites when two adjacent sequences are present, as opposed to a single site alone.Figure 2.


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)

Competition of EbfC binding to probe b-WT by the non-specific competitor poly-dI-dC. EMSA included 100 pM b-WT DNA, and lanes 2–10 contained 1 μM EbfC; also contained either 0, 1, 5, 10, 100, 500, 1000, 5000 or 25 000 pg/μl poly-dI-dC, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665219&req=5

Figure 3: Competition of EbfC binding to probe b-WT by the non-specific competitor poly-dI-dC. EMSA included 100 pM b-WT DNA, and lanes 2–10 contained 1 μM EbfC; also contained either 0, 1, 5, 10, 100, 500, 1000, 5000 or 25 000 pg/μl poly-dI-dC, respectively.
Mentions: Initially, mutants of PerpAB were produced in which either site I, site II or both sites I and II were changed to 5′-CACAACA-3′, all in the context of otherwise normal PerpAB DNA sequences (Figure 1). EMSA using a probe with the wild-type PerpAB sequence, b-WT, yielded two observable DNA–EbfC complexes, with the slower mobility (upper) complex having a higher affinity interaction (Figure 2A). Probes that contain only one TGTA/TACA palindrome (b-C1 and b-C2) exhibited the same two EbfC–DNA complexes, but at decreased relative levels (Figure 3A). The signal strengths of the slower mobility complexes for EbfC binding to either probes b-C1 or b-C2 were approximately one half the value for b-WT. There were no apparent differences in EbfC binding to DNA probes that contained only wild type site I or site II, indicating relatively equal affinities for both sites. However, the mutation of both TGTA/TACA palindromes in probe b-C1/2 dramatically reduced EbfC binding (Figure 2A). These results confirm that EbfC preferentially binds the previously identified palindromic sequence and demonstrate that the apparent binding affinity when two sites are present is greater than when DNA contains only one site. This was likely due to the higher relative concentration of high-affinity binding sites when two adjacent sequences are present, as opposed to a single site alone.Figure 2.

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