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.
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.
Pérez-Marín
Mari Cruz
MC
Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Murcia 30071, Spain.
López-Rubio
Jose Juan
JJ
Murillo
Francisco Jose
FJ
Elías-Arnanz
Montserrat
M
Padmanabhan
S
S
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
D001426:Bacterial Proteins; C091094:CarS protein, Myxococcus xanthus; D012097:Repressor Proteins; D014157:Transcription Factors; D002338:Carotenoids; D004247:DNA; D012321:DNA-Directed RNA Polymerases