| Co-Investigator(Kenkyū-buntansha) |
KASHIWAGI Keiko Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80169424)
KAKINUMA Yoshimi Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80134394)
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| Research Abstract |
1.PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system. The crystal structure of PotD in complex with spermidine has been solved at 2.5-* resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. Spermidine binding sites on PotD were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine, and that Glu-171, Trp-225, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids l
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isted above.
2.Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) was shown to occur mainly at the level of translation by measuring OppA synthesis and its mRNA level. Several artificial oppA genes were constructed by site-directed mutagenesis. These synthesize different kinds of OppA mRNAs : mRNAs differing in the size of 5'-untranslated region (5'-UTR) ; mRNAs having the Shine-Dalgarno (SD) sequence in a different position ; mRNAs having dirrerent secondary structure in the region of the SD sequence ; and fusion mRNAs consisting of the 5'-UTR of OppA mRNA and the open reading frame of beta-galactosidase. By measuring the synthesis of OppA or beta-galactosidase from these mRANs, we found that the 171-nucleotide 5'-UTR and 145 nucleotides of the ORF OppA mRNA are involved in the polyamine stimulation of OppA synthesis. When the secondary structure of the above region of OppA mRNA was analyzed by optimal computer folding, it was shown that the degree of polyamine stimulation of OppA protein synthesis was dependent on the structure of the SD sequence in addition to its position. Loose base pairing of the SD sequence with other regions of the mRNA caused strong polyamine stimulation, while intense base pairing of the SD sequence with other regions of the mRNA resulted in insignificant or weak polyamine stimulation. Less
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