| The interior of a cell is a crowded and constantly fluctuating environment, where DNA and other biomolecules are highly constrained and subject to various kinds of mechanical forces. To unravel the role of mechanics in gene regulation, it is necessary to quantitatively understand the effects of mechanical tension and constraints on protein-mediated DNA looping, which is a ubiquitous theme in the regulation of the expression of prokaryotic and eukaryotic genes.;We have used the lac system in Escherichia coli as the model system to study how mechanical tension and constraints affect the formation and breakdown of regulatory protein-DNA complexes. The lac repressor-mediated DNA loop, which is formed when a lac repressor protein binds to two operator sites on a DNA molecule simultaneously, is the paradigm for protein-mediated DNA looping and is crucial to the repression of the lac genes.;To study the effects of mechanical constraints on the elasticity of DNA, we have developed the constant-force axial optical tweezers to manipulate submicron DNA molecules that are as short as ∼250 bp in length. The force-extension curves of short DNA molecules measured using the optical tweezers show that, because of the entropic boundary effects, the persistence length of a DNA molecule is contour length-dependent and that the excluded-volume force is significant when the molecule is short. In addition, by measuring the formation and breakdown of lac repressor-mediated DNA loops under static tension and fluctuating forces respectively, we have shown that the loop formation rate is sensitive to static tension on the order of only a hundred femtonewtons and to fluctuations of only a fraction of kBT. The loop disruption rate, however, is found to be insensitive to either small static tension or small fluctuations. Moreover, our data show that the sensitivity of the loop formation rate to fluctuations is insensitive to the mean applied tension in the DNA. Our findings suggest not only that tension could be used as a means of regulating the gene transcription but that the hypothetical genetic switch can function robustly even in a noisy in vivo environment. |
