title: Arginine operator binding by heterologous and chimeric ArgR repressors from Escherichia coli and Bacillus stearothermophilus.
abstract: Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the alpha4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the alpha4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.
authors.affiliation: Laboratoire de Biotechnologie, FRE CNRS 2230, Unité Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, 44322 Nantes. IFR 26, INSERM, 44035 Nantes, France.
authors.forename: Anahit
authors.initials: A
authors.lastname: Ghochikyan
authors.forename: Iovka Miltcheva
authors.initials: IM
authors.lastname: Karaivanova
authors.forename: Michèle
authors.initials: M
authors.lastname: Lecocq
authors.forename: Patricia
authors.initials: P
authors.lastname: Vusio
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Arnaud
authors.forename: Marina
authors.initials: M
authors.lastname: Snapyan
authors.forename: Pierre
authors.initials: P
authors.lastname: Weigel
authors.forename: Laetitia
authors.initials: L
authors.lastname: Guével
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.forename: Vehary
authors.initials: V
authors.lastname: Sakanyan
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001120:Arginine