Limits...
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.

Show MeSH

Related in: MedlinePlus

Dimer–dimer interactions and tetrameric model. (a) Two dimers are present in the asymmetric unit of the C2 crystal and are shown here as ribbon diagrams with their respective DNA operators. (b) An orthogonal view of the model in (a). (c) A detailed view of the interacting residues observed between chain B of one dimer and chain F of the adjacent dimer. There is also a contact from chain B of one dimer to the phosphate group of the DNA bound to the adjacent dimer (Lys87–P12). (d) Based on these contacts, a 12 bp spacer was modelled in to form a loop. The DNA is rendered as a space-filling cartoon in the same orientation as in (b), with the crystallographic model in dark orange/yellow and the predicted location of the 12 bp spacer in light orange/yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4321490&req=5

fig7: Dimer–dimer interactions and tetrameric model. (a) Two dimers are present in the asymmetric unit of the C2 crystal and are shown here as ribbon diagrams with their respective DNA operators. (b) An orthogonal view of the model in (a). (c) A detailed view of the interacting residues observed between chain B of one dimer and chain F of the adjacent dimer. There is also a contact from chain B of one dimer to the phosphate group of the DNA bound to the adjacent dimer (Lys87–P12). (d) Based on these contacts, a 12 bp spacer was modelled in to form a loop. The DNA is rendered as a space-filling cartoon in the same orientation as in (b), with the crystallographic model in dark orange/yellow and the predicted location of the 12 bp spacer in light orange/yellow.

Mentions: One interesting possibility is that the two dimeric complexes found in the asymmetric unit of the C2 crystal form of the complex (Figs. 7 ▶a and 7 ▶b) represent such an interaction but lacking a covalently linked DNA spacer. The two dimeric complexes in the asymmetric unit of the C2 crystal (Figs. 7 ▶a and 7 ▶b) are held together by protein–protein interactions between adjacent dimers (Fig. 7 ▶c). Following the notation used in the PDB (entry 4jqd), the dimer at the first site is represented by protein subunits A and B and the two DNA strands in the complex are labelled G and H. Likewise, the subunits of the second complex are labelled E and F and the associated DNA strands C and D. There are clear contacts between adjacent protein dimers in this tetrameric assembly, including a number of ion-pair interactions, in which Asn90 and Glu83 of subunit B in one dimer contact Glu48 and Arg34 (respectively) of subunit F in the adjacent protein dimer. In addition, Lys40 of subunit A is in proximity to Glu83 of subunit F, and these two residues may also interact. There are also potential contacts between a protein subunit of one dimer and the DNA bound to the second dimer; for example, the interaction of Lys87 (subunit B) with a phosphate of the DNA (strand H). We note that three of these interacting residues (Glu83, Lys87 and Asn90) are located in helix 7 at the C-terminus of the protein. This C-terminal region of the amino-acid sequence is rich in basic amino acids (seven from 13 residues, including three arginines and four lysines). This helix is not found in typical C-proteins, suggesting that it has a unique function that may promote protein–protein and protein–DNA interactions to stabilize the tetrameric complex.


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)

Dimer–dimer interactions and tetrameric model. (a) Two dimers are present in the asymmetric unit of the C2 crystal and are shown here as ribbon diagrams with their respective DNA operators. (b) An orthogonal view of the model in (a). (c) A detailed view of the interacting residues observed between chain B of one dimer and chain F of the adjacent dimer. There is also a contact from chain B of one dimer to the phosphate group of the DNA bound to the adjacent dimer (Lys87–P12). (d) Based on these contacts, a 12 bp spacer was modelled in to form a loop. The DNA is rendered as a space-filling cartoon in the same orientation as in (b), with the crystallographic model in dark orange/yellow and the predicted location of the 12 bp spacer in light orange/yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Dimer–dimer interactions and tetrameric model. (a) Two dimers are present in the asymmetric unit of the C2 crystal and are shown here as ribbon diagrams with their respective DNA operators. (b) An orthogonal view of the model in (a). (c) A detailed view of the interacting residues observed between chain B of one dimer and chain F of the adjacent dimer. There is also a contact from chain B of one dimer to the phosphate group of the DNA bound to the adjacent dimer (Lys87–P12). (d) Based on these contacts, a 12 bp spacer was modelled in to form a loop. The DNA is rendered as a space-filling cartoon in the same orientation as in (b), with the crystallographic model in dark orange/yellow and the predicted location of the 12 bp spacer in light orange/yellow.
Mentions: One interesting possibility is that the two dimeric complexes found in the asymmetric unit of the C2 crystal form of the complex (Figs. 7 ▶a and 7 ▶b) represent such an interaction but lacking a covalently linked DNA spacer. The two dimeric complexes in the asymmetric unit of the C2 crystal (Figs. 7 ▶a and 7 ▶b) are held together by protein–protein interactions between adjacent dimers (Fig. 7 ▶c). Following the notation used in the PDB (entry 4jqd), the dimer at the first site is represented by protein subunits A and B and the two DNA strands in the complex are labelled G and H. Likewise, the subunits of the second complex are labelled E and F and the associated DNA strands C and D. There are clear contacts between adjacent protein dimers in this tetrameric assembly, including a number of ion-pair interactions, in which Asn90 and Glu83 of subunit B in one dimer contact Glu48 and Arg34 (respectively) of subunit F in the adjacent protein dimer. In addition, Lys40 of subunit A is in proximity to Glu83 of subunit F, and these two residues may also interact. There are also potential contacts between a protein subunit of one dimer and the DNA bound to the second dimer; for example, the interaction of Lys87 (subunit B) with a phosphate of the DNA (strand H). We note that three of these interacting residues (Glu83, Lys87 and Asn90) are located in helix 7 at the C-terminus of the protein. This C-terminal region of the amino-acid sequence is rich in basic amino acids (seven from 13 residues, including three arginines and four lysines). This helix is not found in typical C-proteins, suggesting that it has a unique function that may promote protein–protein and protein–DNA interactions to stabilize the tetrameric complex.

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