title: The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein.
abstract: Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.
authors.affiliation: Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Murcia 30071, Spain.
authors.forename: Mari Cruz
authors.initials: MC
authors.lastname: Pérez-Marín
authors.forename: Jose Juan
authors.initials: JJ
authors.lastname: López-Rubio
authors.forename: Francisco Jose
authors.initials: FJ
authors.lastname: Murillo
authors.forename: Montserrat
authors.initials: M
authors.lastname: Elías-Arnanz
authors.forename: S
authors.initials: S
authors.lastname: Padmanabhan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002338:Carotenoids / Q000096:biosynthesis*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016941:Myxococcus xanthus / Q000235:genetics*; D009875:Operator Regions, Genetic / Q000235:genetics*; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C091094:CarS protein, Myxococcus xanthus; D012097:Repressor Proteins; D014157:Transcription Factors; D002338:Carotenoids; D004247:DNA; D012321:DNA-Directed RNA Polymerases