title: Operator design and mechanism for CarA repressor-mediated down-regulation of the photoinducible carB operon in Myxococcus xanthus.
abstract: The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.
authors.affiliation: Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Murcia 30071.
authors.forename: José Juan
authors.initials: JJ
authors.lastname: López-Rubio
authors.forename: S
authors.initials: S
authors.lastname: Padmanabhan
authors.forename: Jose María
authors.initials: JM
authors.lastname: Lázaro
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Murillo
authors.forename: Montserrat
authors.initials: M
authors.lastname: Elías-Arnanz
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002338:Carotenoids / Q000096:biosynthesis*; D018983:DNA Footprinting; D015536:Down-Regulation; D015964:Gene Expression Regulation, Bacterial; D008027:Light; D008969:Molecular Sequence Data; D016941:Myxococcus xanthus / Q000235:genetics* / Q000378:metabolism; D009875:Operator Regions, Genetic; D009876:Operon; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D002338:Carotenoids