title: Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis.
abstract: sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5'-TTTGTTCTCTAAAGAGAACTTA-3', containing a pair of SinR consensus sequences in inverted orientation with a K(d) of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which "repurposes" SinR as a repressor of autolysin and motility genes, are discussed.
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK.
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Colledge
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Fogg
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Levdikov
authors.forename: Andrew
authors.initials: A
authors.lastname: Leech
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Dodson
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Wilkinson
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D002845:Chromatography; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D015964:Gene Expression Regulation, Bacterial; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C097712:SinI protein, Bacillus subtilis; C076733:FlaD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase