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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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Gel shift assays carried out using purified CprB and radiolabeled DNA. (A) Result of CprB with 22-mer CS complex formation. (B) Multiple sequence alignment of the upstream regions of cprA (25-bp) and cprB (27-bp) with CS; asterisk indicates the bases conserved in all three sequences and colon indicates the bases conserved in any two sequences aligned. (C–E) The EMSA results showing the CprB–DNA complex formation of purified CprB with longer stretch, 59-mer of upstream sequence (−58 to 0) from cprB-ATG, shorter sequence, 27-mer (−47 to −21) OPB and the upstream sequence, 25-mer (−44 to −20) of cprA-ATG, respectively. The final concentration of CprB is indicated above each lane. The band corresponding to CprB–DNA (complex) and free DNA are indicated. Lane highlighted using asterisk in (D) has excess of cold OPB DNA along with the reaction mixture. All the concentrations are mentioned in micromolar.
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Figure 3: Gel shift assays carried out using purified CprB and radiolabeled DNA. (A) Result of CprB with 22-mer CS complex formation. (B) Multiple sequence alignment of the upstream regions of cprA (25-bp) and cprB (27-bp) with CS; asterisk indicates the bases conserved in all three sequences and colon indicates the bases conserved in any two sequences aligned. (C–E) The EMSA results showing the CprB–DNA complex formation of purified CprB with longer stretch, 59-mer of upstream sequence (−58 to 0) from cprB-ATG, shorter sequence, 27-mer (−47 to −21) OPB and the upstream sequence, 25-mer (−44 to −20) of cprA-ATG, respectively. The final concentration of CprB is indicated above each lane. The band corresponding to CprB–DNA (complex) and free DNA are indicated. Lane highlighted using asterisk in (D) has excess of cold OPB DNA along with the reaction mixture. All the concentrations are mentioned in micromolar.

Mentions: Sugiyama et al. in 1998 have demonstrated that CprB binds to a 22-bp imperfect palindromic CS that has been established to bind the ArpA of S. griseus (46). We employed EMSA studies to confirm and estimate the DNA binding affinity of CprB toward the CS and the values were found to be in the range of 350–400 nM shown in Figure 3A. Although this DNA sequence was instrumental in understanding the conformational changes via the CprB–CS complex structure, the identity of the cognate DNA that CprB regulates in S. coelicolor A3(2) is not known till date. Therefore, to identify the biologically relevant DNA sequence recognized by CprB in its parent organism, we performed a genome-wide search of S. coelicolor A3(2) by using CS as the search string. The search identified the upstream regions of cprA-ATG and cprB-ATG to be the most similar in sequence (Figure 3B and Supplementary Figure S1). To confirm the binding to these identified sequences, firstly a 59-bp oligonucleotide sequence from −58 to 0 region, upstream of cprB-ATG was synthesized. A strongly retarded band of the CprB–DNA complex was observed. The results, as depicted in Figure 3C, show that CprB binds to the 59-bp cprB-ATG DNA with a Kd of ∼400 nM. Two shorter sequences of 27-bp length were subsequently constructed from the 59-bp DNA and it was demonstrated that one of the sequences does not show significant binding to CprB and has very low similarity with the CS. The other 27-bp sequence from −47 to −21 region of the cprB-ATG (OPB) exhibits Kd in the range of 250–300 nM (Figure 3D). A similar trend was observed for the binding of CprB with the cprA-ATG upstream sequence from −44 to −20 (Figure 3E). Moreover, to determine the appropriate length of the sequence sufficient for effective interaction of CprB with DNA, both shorter and longer sequences of DNA were synthesized (Supplementary Figure S1a and b). It was concluded that a longer fragment of DNA is not necessary for enhancement of the binding ability of CprB towards DNA. Nevertheless, it was envisioned that these longer sequences of DNA might favor nucleation for effective crystallization of the complex. A detailed list of all DNA sequences analyzed is presented in Supplementary Table S1. Affinity of CprB with the promoters of scbR (Figure 4A) and the cryptic type I polyketide gene cluster, kasO [kasOB (Figure 4B) and kasOA (Figure 4C)], both of which are regulated by ScbR, were also tested. Results reveal that CprB binds to these sequences with Kd ranging from 0.75 to 1.5 μM, which is around three-fold lower than that observed for the OPB sequence. These results indicate that CprB binds to its own upstream sequence with much greater affinity than the scbR and kasO promoters.


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)

Gel shift assays carried out using purified CprB and radiolabeled DNA. (A) Result of CprB with 22-mer CS complex formation. (B) Multiple sequence alignment of the upstream regions of cprA (25-bp) and cprB (27-bp) with CS; asterisk indicates the bases conserved in all three sequences and colon indicates the bases conserved in any two sequences aligned. (C–E) The EMSA results showing the CprB–DNA complex formation of purified CprB with longer stretch, 59-mer of upstream sequence (−58 to 0) from cprB-ATG, shorter sequence, 27-mer (−47 to −21) OPB and the upstream sequence, 25-mer (−44 to −20) of cprA-ATG, respectively. The final concentration of CprB is indicated above each lane. The band corresponding to CprB–DNA (complex) and free DNA are indicated. Lane highlighted using asterisk in (D) has excess of cold OPB DNA along with the reaction mixture. All the concentrations are mentioned in micromolar.
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Related In: Results  -  Collection

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Figure 3: Gel shift assays carried out using purified CprB and radiolabeled DNA. (A) Result of CprB with 22-mer CS complex formation. (B) Multiple sequence alignment of the upstream regions of cprA (25-bp) and cprB (27-bp) with CS; asterisk indicates the bases conserved in all three sequences and colon indicates the bases conserved in any two sequences aligned. (C–E) The EMSA results showing the CprB–DNA complex formation of purified CprB with longer stretch, 59-mer of upstream sequence (−58 to 0) from cprB-ATG, shorter sequence, 27-mer (−47 to −21) OPB and the upstream sequence, 25-mer (−44 to −20) of cprA-ATG, respectively. The final concentration of CprB is indicated above each lane. The band corresponding to CprB–DNA (complex) and free DNA are indicated. Lane highlighted using asterisk in (D) has excess of cold OPB DNA along with the reaction mixture. All the concentrations are mentioned in micromolar.
Mentions: Sugiyama et al. in 1998 have demonstrated that CprB binds to a 22-bp imperfect palindromic CS that has been established to bind the ArpA of S. griseus (46). We employed EMSA studies to confirm and estimate the DNA binding affinity of CprB toward the CS and the values were found to be in the range of 350–400 nM shown in Figure 3A. Although this DNA sequence was instrumental in understanding the conformational changes via the CprB–CS complex structure, the identity of the cognate DNA that CprB regulates in S. coelicolor A3(2) is not known till date. Therefore, to identify the biologically relevant DNA sequence recognized by CprB in its parent organism, we performed a genome-wide search of S. coelicolor A3(2) by using CS as the search string. The search identified the upstream regions of cprA-ATG and cprB-ATG to be the most similar in sequence (Figure 3B and Supplementary Figure S1). To confirm the binding to these identified sequences, firstly a 59-bp oligonucleotide sequence from −58 to 0 region, upstream of cprB-ATG was synthesized. A strongly retarded band of the CprB–DNA complex was observed. The results, as depicted in Figure 3C, show that CprB binds to the 59-bp cprB-ATG DNA with a Kd of ∼400 nM. Two shorter sequences of 27-bp length were subsequently constructed from the 59-bp DNA and it was demonstrated that one of the sequences does not show significant binding to CprB and has very low similarity with the CS. The other 27-bp sequence from −47 to −21 region of the cprB-ATG (OPB) exhibits Kd in the range of 250–300 nM (Figure 3D). A similar trend was observed for the binding of CprB with the cprA-ATG upstream sequence from −44 to −20 (Figure 3E). Moreover, to determine the appropriate length of the sequence sufficient for effective interaction of CprB with DNA, both shorter and longer sequences of DNA were synthesized (Supplementary Figure S1a and b). It was concluded that a longer fragment of DNA is not necessary for enhancement of the binding ability of CprB towards DNA. Nevertheless, it was envisioned that these longer sequences of DNA might favor nucleation for effective crystallization of the complex. A detailed list of all DNA sequences analyzed is presented in Supplementary Table S1. Affinity of CprB with the promoters of scbR (Figure 4A) and the cryptic type I polyketide gene cluster, kasO [kasOB (Figure 4B) and kasOA (Figure 4C)], both of which are regulated by ScbR, were also tested. Results reveal that CprB binds to these sequences with Kd ranging from 0.75 to 1.5 μM, which is around three-fold lower than that observed for the OPB sequence. These results indicate that CprB binds to its own upstream sequence with much greater affinity than the scbR and kasO promoters.

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