Structure and function of a TetR family transcriptional regulator, SbtR, from <i>thermus thermophilus</i> HB8

Agari, Yoshihiro; Sakamoto, Keiko; Kuramitsu, Seiki; Shinkai, Akeo

doi:10.1002/prot.24266

Cited by 11 publications

(11 citation statements)

References 40 publications

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“…In general, the LH spiral disulfide bonds are known to confer structural stability (55–57). Similar scenario was also observed in another TetR-FTR protein, SbtR ( Thermus thermophilus HB8) where the disulfide bond possess a LH spiral geometry and partakes in stabilizing the dimeric unit (57). To shed light on the role played by the disulfide bond in CprB, cysteine 159 was mutated to serine.…”

Section: Resultssupporting

confidence: 63%

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

Bhukya

Bhujbalrao

Bitra

et al. 2014

Nucleic Acids Research

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Antibiotic production and resistance pathways in Streptomyces are dictated by the interplay of transcriptional regulatory proteins that trigger downstream responses via binding to small diffusible molecules. To decipher the mode of DNA binding and the associated allosteric mechanism in the sub-class of transcription factors that are induced by γ-butyrolactones, we present the crystal structure of CprB in complex with the consensus DNA element to a resolution of 3.25 Å. 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. The crystal structure revealed that, CprB is bound to DNA as a dimer of dimers with the mode of binding being analogous to the broad spectrum multidrug transporter protein QacR from the antibiotic resistant strain Staphylococcus aureus. It was demonstrated that the CprB displays a cooperative mode of DNA binding, following a clamp and click model. 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).

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Section: Resultssupporting

confidence: 63%

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

Bhukya

Bhujbalrao

Bitra

et al. 2014

Nucleic Acids Research

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“…These data strongly suggest that the TTHA1315 and TTHA1316 genes may be regulated by both the TTHA0167 and TTHB023 transcriptional repressors, requiring multiple effectors to be present to permit their maximal expression in vivo. This co-ordinate regulation may also help explain why previous investigators did not report TTHA1315 and TTHA1316 being regulated by TTHA0167 in vivo, even though they well recognized TTHA0167 binding to these gene promoters in genomic selection experiments [22]. We were able to confirm this using publicly available microarray data and GEO2R, finding that TTHA1315 and TTHA1316 are not among the top 250 genes whose expression is affected by TTHA0167 deletion (Table S3).…”

Section: Discussionsupporting

confidence: 66%

“…In the case of TTHA1315/16, we believe multiple transcriptional repressors may be involved in their regulation. In fact, we had previously shown that these genes are likely targets of another T. thermophilus HB8 TetR-family transcriptional repressor, TTHA0167, otherwise known as SbtR [4,22]. We found that the consensus sequence for TTHA0167 is 5 -TGACYrnnyRGTCA-3 , which can be accommodated by the central 14 nucleotides of the TTHB023 consensus sequence in many cases.…”

Section: Discussionmentioning

confidence: 97%

General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

Cox

Dyke

2020

Biomolecules

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Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation.

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“…As TetR proteins normally exist as homodimers under physiological conditions, the location of C164 near the C-terminus suggests that it could participate in disulfide bond formation. A 2.05 Å crystal structure for SbtR presently exists (RCSB PDB 3VUQ) [ 24 ]. Its asymmetric unit is comprised of two SbtR homodimers.…”

Section: Resultsmentioning

confidence: 99%

“…used four rounds of SELEX with a C-terminal his6-tagged SbtR and PCR-generated inserts from a T . thermophilus genomic library to identify DNAs containing SbtR binding sites [ 24 ]. Of the 46 clones sequenced, 13 contained bona fide T .…”

Section: Discussionmentioning

confidence: 99%

Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA

et al. 2016

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One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles.

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Structure and function of a TetR family transcriptional regulator, SbtR, from thermus thermophilus HB8

Cited by 11 publications

References 40 publications

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

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

General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA

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