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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.

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.

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Overall structure of the C.Csp231I–DNA complex. (a) The protein dimer (cyan and violet subunits) bound to a DNA duplex (orange). The specific DNA-recognition sites (yellow) are located on both sides of the central pentanucleotide spacer. (b) Orthogonal view of the structure in (a).
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fig2: Overall structure of the C.Csp231I–DNA complex. (a) The protein dimer (cyan and violet subunits) bound to a DNA duplex (orange). The specific DNA-recognition sites (yellow) are located on both sides of the central pentanucleotide spacer. (b) Orthogonal view of the structure in (a).

Mentions: The overall structure of the complex (Fig. 2 ▶) consists of a C-protein dimer bound to a DNA duplex. The structure of the free C.Csp231I protein dimer contains seven helices, as found in the free protein (McGeehan et al., 2011 ▶), but with subtle conformational differences in the DNA-bound form of the protein. In the complex, each subunit interacts with the DNA by inserting recognition helix 3 (residues 28–40) of the classical helix–turn–helix motif into the major groove of the DNA either side of the central GAAAA motif. Superposition of monomer-to-monomer main-chain atoms reveals only minor differences when comparing subunits within the dimer. The maximum displacement between the main-chain atoms of separate monomers is confined to the C-terminal region (residues 86–95) of the protein (1.1 Å). This difference reflects conformational flexibility in this region of the protein, which has elevated values of crystallographic temperature (B) factors (see Supplementary Fig. S2). The observed flexibility of the C-terminal domain is similar in magnitude to that of the free protein structure (McGeehan et al., 2011 ▶).


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)

Overall structure of the C.Csp231I–DNA complex. (a) The protein dimer (cyan and violet subunits) bound to a DNA duplex (orange). The specific DNA-recognition sites (yellow) are located on both sides of the central pentanucleotide spacer. (b) Orthogonal view of the structure in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Overall structure of the C.Csp231I–DNA complex. (a) The protein dimer (cyan and violet subunits) bound to a DNA duplex (orange). The specific DNA-recognition sites (yellow) are located on both sides of the central pentanucleotide spacer. (b) Orthogonal view of the structure in (a).
Mentions: The overall structure of the complex (Fig. 2 ▶) consists of a C-protein dimer bound to a DNA duplex. The structure of the free C.Csp231I protein dimer contains seven helices, as found in the free protein (McGeehan et al., 2011 ▶), but with subtle conformational differences in the DNA-bound form of the protein. In the complex, each subunit interacts with the DNA by inserting recognition helix 3 (residues 28–40) of the classical helix–turn–helix motif into the major groove of the DNA either side of the central GAAAA motif. Superposition of monomer-to-monomer main-chain atoms reveals only minor differences when comparing subunits within the dimer. The maximum displacement between the main-chain atoms of separate monomers is confined to the C-terminal region (residues 86–95) of the protein (1.1 Å). This difference reflects conformational flexibility in this region of the protein, which has elevated values of crystallographic temperature (B) factors (see Supplementary Fig. S2). The observed flexibility of the C-terminal domain is similar in magnitude to that of the free protein structure (McGeehan et al., 2011 ▶).

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