Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping.
Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.
Normanno
Davide
D
LENS, European Laboratory for Non-linear Spectroscopy, Università degli Studi di Firenze, Via N. Carrara 1, I-50019 Sesto Fiorentino (FI), Italy. dnormanno@ibec.pcb.ub.es
Vanzi
Francesco
F
Pavone
Francesco Saverio
FS
D001426:Bacterial Proteins / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D005821:Genetic Techniques; D007700:Kinetics; D056968:Lac Repressors; D008863:Microspheres; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D056968:Lac Repressors; D012097:Repressor Proteins