title: High-mobility-group a-like CarD binds to a DNA site optimized for affinity and position and to RNA polymerase to regulate a light-inducible promoter in Myxococcus xanthus.
abstract: The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (P(QRS)), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P(QRS) had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P(QRS) activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD(Ad)-CarG(Ad)). CarD(Ad)-CarG(Ad) is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD(Ad), whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.
authors.affiliation: Departamento de Genética y Microbiología, Área de Genética (Unidad Asociada al Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas), Facultad de Biología, Universidad de Murcia, Murcia, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Heras
authors.forename: Javier
authors.initials: J
authors.lastname: Abellón-Ruiz
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Murillo
authors.forename: S
authors.initials: S
authors.lastname: Padmanabhan
authors.forename: Montserrat
authors.initials: M
authors.lastname: Elías-Arnanz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008027:Light; D008969:Molecular Sequence Data; D016941:Myxococcus xanthus / Q000235:genetics* / Q000528:radiation effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C100589:CarD protein, Myxococcus xanthus; D004269:DNA, Bacterial; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit