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Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression.

Shevtsov MB, Streeter SD, Thresh SJ, Swiderska A, McGeehan JE, Kneale GG - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon.We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC.An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA.

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Affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY, England.

ABSTRACT
In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

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Structural distortions in bound DNA. (a) The local bend angle for 21-mer OL (blue) and OR (red) operators between adjacent base pairs (calculated as the angle formed between the normals of adjacent base pairs) is greatest at the central spacer sequence. Their sequences are shown below with the inverted repeats underlined. (b) Graphical representation of duplex bending. The overall DNA bend angles are 39 and 43° for the OL and OR duplexes, respectively
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fig4: Structural distortions in bound DNA. (a) The local bend angle for 21-mer OL (blue) and OR (red) operators between adjacent base pairs (calculated as the angle formed between the normals of adjacent base pairs) is greatest at the central spacer sequence. Their sequences are shown below with the inverted repeats underlined. (b) Graphical representation of duplex bending. The overall DNA bend angles are 39 and 43° for the OL and OR duplexes, respectively

Mentions: The DNA duplex in both complexes is significantly distorted from canonical B-form DNA (Fig. 4 ▶), with a bend of 39 and 43° for the complexes with OL and OR, respectively, similar to the value (41°) observed in complexes of C.Esp231I with the OL operator (McGeehan et al., 2012 ▶). The bend angle induced in DNA when bound to the related C.EcoO109, as estimated from gel assays, was reported to be 54° (Kita et al., 2002 ▶), although the results are not strictly comparable as they were obtained using different techniques.


Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression.

Shevtsov MB, Streeter SD, Thresh SJ, Swiderska A, McGeehan JE, Kneale GG - Acta Crystallogr. D Biol. Crystallogr. (2015)

Structural distortions in bound DNA. (a) The local bend angle for 21-mer OL (blue) and OR (red) operators between adjacent base pairs (calculated as the angle formed between the normals of adjacent base pairs) is greatest at the central spacer sequence. Their sequences are shown below with the inverted repeats underlined. (b) Graphical representation of duplex bending. The overall DNA bend angles are 39 and 43° for the OL and OR duplexes, respectively
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321490&req=5

fig4: Structural distortions in bound DNA. (a) The local bend angle for 21-mer OL (blue) and OR (red) operators between adjacent base pairs (calculated as the angle formed between the normals of adjacent base pairs) is greatest at the central spacer sequence. Their sequences are shown below with the inverted repeats underlined. (b) Graphical representation of duplex bending. The overall DNA bend angles are 39 and 43° for the OL and OR duplexes, respectively
Mentions: The DNA duplex in both complexes is significantly distorted from canonical B-form DNA (Fig. 4 ▶), with a bend of 39 and 43° for the complexes with OL and OR, respectively, similar to the value (41°) observed in complexes of C.Esp231I with the OL operator (McGeehan et al., 2012 ▶). The bend angle induced in DNA when bound to the related C.EcoO109, as estimated from gel assays, was reported to be 54° (Kita et al., 2002 ▶), although the results are not strictly comparable as they were obtained using different techniques.

Bottom Line: In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon.We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC.An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY, England.

ABSTRACT
In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

Show MeSH
Related in: MedlinePlus