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Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2).

Bhukya H, Bhujbalrao R, Bitra A, Anand R - Nucleic Acids Res. (2014)

Bottom Line: Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove.Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator.Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.

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EMSA studies of mutant CprB carried out with cprB upstream sequence (OPB). (A) Backbone interacting residue S33 was mutated to alanine residue in native CprB; (B) base interacting residue K43 was mutated to alanine residue; and (C) double mutation of S33 and K43 residues to alanine residue. Single mutations have negligible effect on DNA binding. Double mutants, however, result in drastic decrease in DNA binding ability. All the concentrations are mentioned in micromolar.
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Figure 6: EMSA studies of mutant CprB carried out with cprB upstream sequence (OPB). (A) Backbone interacting residue S33 was mutated to alanine residue in native CprB; (B) base interacting residue K43 was mutated to alanine residue; and (C) double mutation of S33 and K43 residues to alanine residue. Single mutations have negligible effect on DNA binding. Double mutants, however, result in drastic decrease in DNA binding ability. All the concentrations are mentioned in micromolar.

Mentions: To decipher the importance of the structural basis of interaction of CprB with the DNA, a couple of variants of CprB were designed via inspection of the CprB–CS complex crystal structure. First, a series of single amino acid mutations in CprB were made. All residues selected for mutagenesis are from the spacer helix α2 and recognition helix α3. Representative mutations disrupting the phosphate backbone interactions constructed were S33A and T31A and the base interacting single mutants engineered were Y47A, F48A and K43A. The mutant proteins were purified and tested for binding with both the OPB and the cprA-ATG upstream sequences. The results of the single point mutation in CprB showed that there is almost no effect on the ability of the protein to bind with DNA (Figure 6A and B). This highlights the importance of the fact that single mutations can be absorbed by this class of proteins without affecting DNA binding ability of CprB. Further, to investigate the ability of the CprB to absorb multiple mutations, a double mutation (S33A, K43A) which disrupts both a phosphate backbone contact and a DNA base interaction was constructed. The double mutant showed a pronounced effect (Figure 6C). We conclude that since the DNA makes several interactions with the four monomers of the protein forming an extensive interface, disruption of a few interactions does not affect DNA binding. On the other hand, the creation of double mutant results in a cumulative reduction of protein–DNA contacts, thereby drastically impairing the DNA binding capability of the protein.


Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2).

Bhukya H, Bhujbalrao R, Bitra A, Anand R - Nucleic Acids Res. (2014)

EMSA studies of mutant CprB carried out with cprB upstream sequence (OPB). (A) Backbone interacting residue S33 was mutated to alanine residue in native CprB; (B) base interacting residue K43 was mutated to alanine residue; and (C) double mutation of S33 and K43 residues to alanine residue. Single mutations have negligible effect on DNA binding. Double mutants, however, result in drastic decrease in DNA binding ability. All the concentrations are mentioned in micromolar.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150764&req=5

Figure 6: EMSA studies of mutant CprB carried out with cprB upstream sequence (OPB). (A) Backbone interacting residue S33 was mutated to alanine residue in native CprB; (B) base interacting residue K43 was mutated to alanine residue; and (C) double mutation of S33 and K43 residues to alanine residue. Single mutations have negligible effect on DNA binding. Double mutants, however, result in drastic decrease in DNA binding ability. All the concentrations are mentioned in micromolar.
Mentions: To decipher the importance of the structural basis of interaction of CprB with the DNA, a couple of variants of CprB were designed via inspection of the CprB–CS complex crystal structure. First, a series of single amino acid mutations in CprB were made. All residues selected for mutagenesis are from the spacer helix α2 and recognition helix α3. Representative mutations disrupting the phosphate backbone interactions constructed were S33A and T31A and the base interacting single mutants engineered were Y47A, F48A and K43A. The mutant proteins were purified and tested for binding with both the OPB and the cprA-ATG upstream sequences. The results of the single point mutation in CprB showed that there is almost no effect on the ability of the protein to bind with DNA (Figure 6A and B). This highlights the importance of the fact that single mutations can be absorbed by this class of proteins without affecting DNA binding ability of CprB. Further, to investigate the ability of the CprB to absorb multiple mutations, a double mutation (S33A, K43A) which disrupts both a phosphate backbone contact and a DNA base interaction was constructed. The double mutant showed a pronounced effect (Figure 6C). We conclude that since the DNA makes several interactions with the four monomers of the protein forming an extensive interface, disruption of a few interactions does not affect DNA binding. On the other hand, the creation of double mutant results in a cumulative reduction of protein–DNA contacts, thereby drastically impairing the DNA binding capability of the protein.

Bottom Line: Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove.Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator.Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.

Show MeSH
Related in: MedlinePlus