Single-Molecule Physiology Under an Optical Microscope:
How Molecular Machines May Work

Kazuhiko Kinosita, Jr. Center for Integrative Bioscience, Okazaki National Research Institutes, Higashiyama 5-1, Myodaiji, Okazaki 444-8585, Japan e-mail: kazuhiko@ims.ac.jp URL: http://www.k2.ims.ac.jp

A single molecule of protein (or RNA) enzyme acts as a machine that carries out a unique function in cellular activities. To elucidate the mechanisms of various molecular machines, we need to observe closely the behaviors of individual molecules, because these machines, unlike man-made machines, operate stochastically and thus cannot be synchronized with each other. By attaching a tag that is huge compared to the size of a molecular machine, or a small tag such as a single fluorophore, we have been able to image the individual behaviors in real time under an optical microscope. Stepping rotation of the central subunit in a single molecule of F₁-ATPase has been videotaped, and now we can discuss its detailed mechanism [1,2]. RNA polymerase has been shown to be a helical motor that precisely tracks the right-handed double helix of DNA [3], whereas myosin V has been shown to proceed as a left-handed spiral around the right-handed double helix of actin [4]. Huge tags such as micron-sized plastic beads also allow the manipulation of individual molecules with, e.g., optical or magnetic tweezers [3-6]. I personally believe that molecular machines operate by changing their conformations. Thus, detection of the conformational changes during function is our prime goal.