Search

Significance Six decades after DNA structure was first revealed, fundamental questions remain open. How is the entwined embrace of double-stranded nucleic acids formed or disrupted? How does the energetics underlying this process influence nucleic-acid processing machineries? By combining simulations and experiments, our work addresses these questions and reveals that asymmetric base-pair dynamics drives the stepwise separation of nucleic acid duplexes, predicts the unwinding efficiency of helicases, and intimately relates the intrinsic dynamics of base pairs to the enzymatic mechanism evolved for their opening. Taken together, our data suggest a layer of regulation of the genetic material encoded in the “unwindability” of the double helix., The opening of a Watson–Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

Funding: This work was supported by the National Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). P.S.P. was supported through a Doctoral fellowship from NSERC and D.A.L was supported through a Fonds de Recherche Santé Québec Senior scholarship. C. Perez-Gonzalez and E. Shaw thank the Engineering and Physical Sciences Research Council (EPSRC) and The University of St Andrews for PhD studentships. Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that preorganizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation. Publisher PDF

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions We thank Dr. Arvydas Ruseckas for helping with fluorescence lifetime measurements and the EPSRC (EP/P030017) for support SI