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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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DNA-binding analysis. EMSAs showing the binding of C.Csp231I to various DNA sequences: (a) native 54 bp (54-1), (b) a 54-mer with a random second site (54-2), (c) a 54-mer with a random spacer (54-3) and (d) a 54 bp DNA fragment lacking the central 6 bp spacer (48-1). Precise sequences of the oligonucleotide duplexes are shown in Fig. 1 ▶. DNA duplexes were incubated at protein (subunit):DNA molar ratios of 0, 1, 2, 4 and 8 in each case. The DNA concentration was maintained at 2 µM throughout.
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fig3: DNA-binding analysis. EMSAs showing the binding of C.Csp231I to various DNA sequences: (a) native 54 bp (54-1), (b) a 54-mer with a random second site (54-2), (c) a 54-mer with a random spacer (54-3) and (d) a 54 bp DNA fragment lacking the central 6 bp spacer (48-1). Precise sequences of the oligonucleotide duplexes are shown in Fig. 1 ▶. DNA duplexes were incubated at protein (subunit):DNA molar ratios of 0, 1, 2, 4 and 8 in each case. The DNA concentration was maintained at 2 µM throughout.

Mentions: EMSA analysis of C-protein binding to the left and right operators showed no differences in affinity between the two sites (data not shown). We thus investigated the interaction of C.Csp231I with longer DNA sequences corresponding to the 54 bp region encompassing the operator sites upstream of the C-gene (Fig. 1 ▶). EMSA experiments using the wild-type 54 bp fragment (Fig. 3 ▶a) revealed a single complex at ratios of up to 2:1 (protein subunits per DNA duplex). At ratios of 4:1 and above a larger complex becomes apparent. These species are most likely to correspond to one and two bound dimers, respectively: one dimer bound at each palindromic recognition site of the DNA. We also looked at binding to an equivalent 54 bp oligonucleotide in which the sequence of the right-hand operator had been randomized (see Fig. 1 ▶). It is clear that mutation of this binding site blocks formation of the second species, suggesting that now only a single dimer binds to the wild-type OL site (Fig. 3 ▶b).


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)

DNA-binding analysis. EMSAs showing the binding of C.Csp231I to various DNA sequences: (a) native 54 bp (54-1), (b) a 54-mer with a random second site (54-2), (c) a 54-mer with a random spacer (54-3) and (d) a 54 bp DNA fragment lacking the central 6 bp spacer (48-1). Precise sequences of the oligonucleotide duplexes are shown in Fig. 1 ▶. DNA duplexes were incubated at protein (subunit):DNA molar ratios of 0, 1, 2, 4 and 8 in each case. The DNA concentration was maintained at 2 µM throughout.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: DNA-binding analysis. EMSAs showing the binding of C.Csp231I to various DNA sequences: (a) native 54 bp (54-1), (b) a 54-mer with a random second site (54-2), (c) a 54-mer with a random spacer (54-3) and (d) a 54 bp DNA fragment lacking the central 6 bp spacer (48-1). Precise sequences of the oligonucleotide duplexes are shown in Fig. 1 ▶. DNA duplexes were incubated at protein (subunit):DNA molar ratios of 0, 1, 2, 4 and 8 in each case. The DNA concentration was maintained at 2 µM throughout.
Mentions: EMSA analysis of C-protein binding to the left and right operators showed no differences in affinity between the two sites (data not shown). We thus investigated the interaction of C.Csp231I with longer DNA sequences corresponding to the 54 bp region encompassing the operator sites upstream of the C-gene (Fig. 1 ▶). EMSA experiments using the wild-type 54 bp fragment (Fig. 3 ▶a) revealed a single complex at ratios of up to 2:1 (protein subunits per DNA duplex). At ratios of 4:1 and above a larger complex becomes apparent. These species are most likely to correspond to one and two bound dimers, respectively: one dimer bound at each palindromic recognition site of the DNA. We also looked at binding to an equivalent 54 bp oligonucleotide in which the sequence of the right-hand operator had been randomized (see Fig. 1 ▶). It is clear that mutation of this binding site blocks formation of the second species, suggesting that now only a single dimer binds to the wild-type OL site (Fig. 3 ▶b).

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