||Studies of transcription factor FMBP-1 derived from silkworm Bombyx mori by using fluorescence correlation spectroscopy
蛍光相関分光法を用いたカイコ Bombyx mori 由来転写因子FMBP-1の研究
Fibroin modulator binding protein 1 (FMBP-1) is a silkworm transcription factor thathas a unique DNA binding domain called the one score and three amino acid peptiderepeat (STPR). Here I used fluorescence correlation spectroscopy (FCS) to analyze thediffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein(EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at thesame developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearlylocalized to cell nuclei. From the FCS analyses, I identified an immobile DNA-boundcomponent and three discernible diffusion components. I also used FCS to observe themovements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simplerexperimental system. Based on previous in vitro observation, I also introduced a singleamino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, Ireplaced the ninth Arg in the third repeat within the STPR domain with Ala. Thismutation completely disrupted the slowest diffusion component as well as the immobilecomponent. The diffusion properties of other FMBP-1 mutants (e.g., mutants withN-terminal or C-terminal truncations) were also analyzed. Based on my observations, Isuggest that the four identifiable movements might correspond to four distinct FMBP-1states: 1) diffusion of free protein, 2) and 3) two types of transient interactions betweenFMBP-1 and chromosomal DNA, and 4) stable binding of FMBP-1 to chromosomalDNA.
Hokkaido University（北海道大学）. 博士(生命科学)