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Importin-[beta] mediates protein transport across the nuclear envelope through the nuclear pore complex (NPC) by interacting with components of the NPC, called nucleoporins, and a small G protein, Ran. Although there is accumulated knowledge on the specific interaction between importin-[beta] and the Phe-Gly (FG) motif in the nucleoporins as well as the effect of RanGTP on this interaction, the molecular mechanism by which importin-[beta] shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-[beta] for the FG motifs and characterized the interaction using a single-molecule force-measurement technique with atomic-force microscopy. The results from a series of importin-[beta] mutants containing amino acid substitutions within the FG-binding pockets demonstrate that the individual FG-binding pockets have different affinities to FG-Nups (Nup62 and Nup153) and different sensitivities to RanGTP; the binding of RanGTP to the amino-terminal domain of importin-[beta] induces the conformational change of the entire molecule and reduces the affinity of some of the pockets but not others. These heterogeneous characteristics of the multiple FG-binding pockets may play an important role in the behavior of importin-[beta] within the NPC. Single-molecule force measurement using the entire molecule of an NPC from a Xenopus oocyte also implies that the reduction of the affinity by RanGTP really occurs at the nucleoplasmic side of the entire NPC. nuclear import | nuclear pore complex | nuclear transport | scanning probe microscopy | single-molecule force measurement

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The [...]

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 [+ or -] 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour. imaging | nucleic acid | restriction-modification enzyme | scanning-probe

The effect of linker histone H1 on the higher-order folding of in vitro-reconstituted nucleosome fibers is examined. A flexible model of chromatin fiber formation is proposed where the mode of the fiber compaction changes depending both on salt environment and linker histone H1.

A replication initiator protein from bacterial mini-F plasmid can shift the conformation of DNA from a supercoiled to a relaxed state without local melting of the DNA strand or a strand break. The protein is also not repeatedly wrapped by DNA.

The covalent bond trapping of protein molecules of GroEL and streptavidin conjugated with quantum dot at the open-ended tip of sharpened multiwalled carbon nanotubes (MWCNTs) through diimide-activated amidation are demonstrated.

Katayama Y, Shige H, Yamamoto A, et al. J Atheroscler Thromb 2004;11:354-357. We have investigated whether the oral administration of vitamin C could prevent smoking-induced acceleration of arterial stiffness in [...]