title: Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Michael W
authors.identifier: http://orcid.org/0000-0003-0306-4002
authors.initials: MW
authors.lastname: Van Dyke
authors.affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Beyer
authors.affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Emily
authors.initials: E
authors.lastname: Clay
authors.affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Kamir J
authors.initials: KJ
authors.lastname: Hiam
authors.affiliation: Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: McMurry
authors.affiliation: Department of Computer Science, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xie
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D063990:Gene Ontology; D058977:Molecular Sequence Annotation; D021141:Nucleic Acid Amplification Techniques; D059372:Nucleotide Motifs; D009876:Operon; D011485:Protein Binding; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015252:Deoxyribonucleases, Type II Site-Specific