lac repressor-DNA interaction kinetics under torsional constraints

_ During my Ph.D., I started being interested in transcription regulation and especially in the relationship between DNA mechanics and protein-DNA interactions. More into the details, I investigated how DNA supercoiling was affecting the interaction kinetics between the DNA and the lac repressor (LacI), a DNA-looping transcription factor regulating lactose digestion in bacteria. In particular, I used a single-molecule assay to observe in real-time the LacI activity and to simultaneously control the degree of supercoiling of the DNA, under very low stretching forces (i.e. in the Entropic regime of the DNA), thanks to a 3D magneto-optical micro-manipulator. The micro-manipulation experiments I performed revealed that the LacI-DNA looping kinetics are modulated by the degree of supercoiling of the DNA and that the LacI-DNA looped complex undergoes to a structural rearrangement, with the LacI forced to assume an “open” conformation, in response to the relative orientation of the lac binding sites along the DNA molecules. These findings corroborate the view that DNA topology plays a key role in modulating the efficiency of transcription repression in the lac system and that DNA supercoiling can indeed represent a powerful message relaying global effects of environmental changes onto specific responses of regulatory systems, provided that those systems are sensitive to supercoiling.

Related publications

Normanno D., Vanzi F., and Pavone F.S., “Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping”. Nucleic Acids Research 36, 8, 2505-2513 (2008)

People involved

Prof. Francesco S. Pavone
Dr. Francesco Vanzi

Institutions & fundings

Department of Physics
University of Florence (Italy)
Schematics of the Tethered-Particle-Motion assay used to measure the LacI looping kinetics

Single DNA molecules torsional manipulation under very low stretching forces

The LacI looping kinetics are modulated by the degree of supercoiling of the DNA

LacI undergoes to a conformational change in response to DNA torsional manipulation