Repression by DNA looping: crystal structure and functional analysis of the global transcriptional regulator SCOC

The global transcriptional regulator ScoC belongs to a group of transition state regulators controlling genes at the transition from exponential growth to stationary phase. Using gel retardation assays, fluorescence resonance energy transfer (FRET) analyses, AFM microscopy, and transcriptional lacZ-fusions in B. subtilis background, we demonstrate that ScoC from Geobacillus stearothermophilus binds to two operator sites in the oppA promoter region, and both binding sites are necessary for high repressive effect.

The 3D structures of ScoC and ScoC complexed with DNA were determined at 2.8 Å resolution, revealing a tetrameric X-shape assembly composed of two dimers. FRET analyses and gel retardation assays indicate that a single tetrameric ScoC binds two operator sites and can induce DNA looping. The initial rate of fluoresce intensity in the presence of increasing concentrations of the ScoC protein can be modeled into a typical Michaelis-Menten kinetics, exhibiting uncompetitive substrate inhibition, suggesting that at high concentrations of ScoC, two ScoC tetramers (rather than a single tetramer) bind to the two operators, thus preventing DNA loop formation. Using AFM we obtained images showing the ScoC induce DNA loops. Specific amino acid replacements in ScoC, which interact with the DNA bases in the wing recognition site and which create hydrogen bonds in the major groove via the recognition helix, resulted in reduced ability of ScoC to bind DNA.

In conclusion, we showed that ScoC can bind to two operator sites in the oppA promoter region so as to allow full repression of transcription, most likely by inducing DNA looping.