Recent advances in single-molecule fluorescence microscope techniques have allowed singlemolecule
sensitivity to probe various protein-DNA interactions, their structural changes, and
fundamental cellular processes in a living cell. In this talk, I will introduce a novel single-molecule
fluorescence microscope technique and single-particle tracking method based on super-resolution
imaging, which were used for investigating cellular dynamics in living cells. Transcription, a process
of mRNA generation by RNA polymerase (RNAP), is highly coupled with translation by ribosome in
bacteria. The effect of the transcription-translation coupling on the transcriptional dynamics and the
localization of genes in a living cell is poorly understood. Here, we directly observe the dynamics
of transcription and the movement of the subcellular localization of genes actively transcribed by
RNAP in living cells at the sub-diffraction limit resolution. The subcellular localizations of the nonmembrane
protein’ genes, actively transcribed by RNAPs, move toward outside nucleoid or to
plasma membrane by the effect of translation by ribosome. The movement of genes by
transcription-translation coupling is general for both E. coli RNAP and T7 RNAP. Our observation
demonstrates how two spatially separated processes of transcription and translation are coupled in
bacteria and the movement of genes by the cooperation between transcription and translation plays
a crucial role in the effective expression of genes in living cells. This work paves the way to observe
individual protein machinery and to reveal fundamental cellular processes in living cells. In the
second part, I will introduce real-time single-protein tracking in a living cell, which was used to
study the interactions of membrane proteins. Finally, I will introduce new single-molecule FRET
technique for observing millisecond conformational change of biomolecules.