Keisuke Fujita

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

Temperature at the focal point of optical trapping beam: evaluation using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy was applied to the evaluation of the local heating at the focal spot of nearinfrared laser for optical trapping. Based on the translational diffusion coefficient of probe dyes at the focal spot in solution, the relation between temperature rise and incident laser power, ΔT/ΔP, were determined for water, ethylene glycol, 1-pentanol, 1-hexanol, 1-octanol, 1-nonanol, and 1-decanol. The value of ΔT/ΔP linearly increased with a/l (a and l is the absorption coefficient and thermal conductivity of solvent, respectively) as predicted by a simple theoretical model.

Single-Molecule Analysis of Actomyosin in the Presence of Osmolyte

Actomyosin is a protein complex composed of myosin and actin, which is well known for being the minimal contractile unit of muscle. The chemical free energy of ATP is converted into mechanical work by the complex, and the single-molecule mechanical properties of myosin are well characterized in vitro. However, the aqueous solution environment in in vitro assay is far from that in cells, where biomolecules are crowded, which influences osmotic pressure, and processes such as folding, and association and diffusion of proteins. Here, to bridge the gap between in vitro and in-cell environment, we observed mechanical motion of actomyosin-V in the presence of the osmolyte sucrose, as a model system. Single-molecule observation of myosin-V motor domains (heads) on actin filament at varying sucrose concentration revealed modulated mechanical elementary processes suggesting increased affinity of heads with actin and more robust force generation possibly accompanied by a sliding motion of myosin head along actin.

Myosin V is a biological Brownian machine

Myosin V is a vesicle transporter that unidirectionally walks along cytoskeletal actin filaments by converting the chemical energy of ATP into mechanical work. Recently, it was found that myosin V force generation is a composition of two processes: a lever-arm swing, which involves a conformational change in the myosin molecule, and a Brownian search-and-catch, which involves a diffusive “search” by the motor domain that is followed by an asymmetric “catch” in the forward actin target such that Brownian motion is rectified. Here we developed a system that combines optical tweezers with DNA nano-material to show that the Brownian search-and-catch mechanism is the energetically dominant process at near stall force, providing 13 kBT of work compared to just 3 kBT by the lever-arm swing. Our result significantly reconsiders the lever-arm swinging model, which assumes the swing dominantly produces work (>10 kBT), and sheds light on the Brownian search-and-catch as a driving process.