The transcriptional repressor TtgV recognizes a complex operator as a tetramer and induces convex DNA bending.
The TtgV repressor belongs to the large but infrequently investigated IclR family of transcriptional regulators. Although members of this family usually exhibit high effector specificity, TtgV possesses multidrug binding properties. The TtgV protein regulates the expression of the ttgGHI operon encoding the main solvent extrusion pump of the extremophile Pseudomonas putida DOT-T1E strain. Here we used a multidisciplinary approach to study the functional oligomeric state of TtgV during repression and derepression events, as well as the molecular basis of TtgV-DNA operator interactions. Analytical ultracentrifugation studies (AUC) show that TtgV is a tetramer in solution and that this oligomeric state does not change in the presence of effectors. We also show that the binding of effectors leads to the dissociation of TtgV as a tetramer from the DNA-TtgV complex. Previous dimethyl sulfate and DNase I footprints revealed that TtgV protected a 42 bp region. Based on AUC, electrophorectic mobility shift assays and isothermal titration calorimetry analyses we show that TtgV recognition specificity is restricted within this operator to a 34-nucleotide stretch and that TtgV may interact with intercalated inverted repeats that share no significant DNA sequence similarities within this short 34-nucleotide segment. Binding stoichiometry is one TtgV tetramer per operator, and affinity for its target DNA is around 200 nM. Circular dichroism analysis reveals that TtgV binding causes DNA distortion and atomic force microscopy imaging of TtgV-DNA operator complexes shows that TtgV induces a 57 degrees convex bend in its operator DNA. We propose that the mechanism of TtgV repression is based on the steric occlusion of the RNA polymerase binding site reinforced by DNA-bending of the ttgV-ttgG promoter region.
Guazzaroni
María-Eugenia
ME
Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Environmental Protection, Granada, Spain.
Krell
Tino
T
Gutiérrez del Arroyo
Paloma
P
Vélez
Marisela
M
Jiménez
Mercedes
M
Rivas
Germán
G
Ramos
Juan L
JL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D009876:Operon; D020836:Protein Structure, Quaternary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D004247:DNA