| Project/Area Number |
01480489
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Hokkaido University |
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Principal Investigator |
OHTSUKA Eiko Hokkaido University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (80028836)
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| Co-Investigator(Kenkyū-buntansha) |
KAMIYA Hiroyuki Hokkaido University, Faculty of Pharmaceutical Sciences, Research Assistant, 薬学部, 教務職員 (10204629)
IWAI Shigenori Hokkaido University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (10168544)
INOUE Hideo Hokkaido University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80088856)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | RNA / hammerhead-type ribozyme / self-cleavage reaction / synthetic oligonucleotide / kinetic parameter / structure analysis / CD spectrum / NMR spectrum / オリゴリボヌクレオチド / リボザイム |
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| Research Abstract |
Self-cleaving RNA found in viruses and viroids is the smallest catalytic RNA molecule named "ribozyme", which requires a "hammerhead" structure for its activity. We have investigated this self-cleavage reaction using chemicallysynthesized oligonucleotides in order to elucidate its mechanism.
First we have constructed a two-stranded ribozyme complex and its mutants with a sequence derived from a satellite DNA transcript from the newt, in order to analyze the self-cleavage reaction, and found the consensus sequence required for the reaction. Then we have measured CD spectra of a three-stranded complex containing 2'-O-methylcytidine at the cleavage site, which prevented the cleavage reaction, to show that formation of the tertiary structure is essential to this reaction. The kinetic parameters of the three-stranded complex were as follows : K_m = 0.53 uM, V_<max> = 0.015 uM^.min^<-1>, and k_<cat> = 0.03 min ^<-1>.
As a dimer structure is reported to be preferential in the case of the hammerhead-type ribozyme containing a unstable stem-loop structure, we have analyzed the monomer/dimer structure of the two-stranded ribozyme complex derived from the newt sequence. The following results demonstrate that this complex exists as a monomer : 1) The free energy calculated from its T_m values was almost the same as that obtained for a monomer model. 2) Its electrophoretic mobility on a gel coincided with the monomer-type three-stranded complex. 3) Its half life in the cleavage reaction was very long, compared with that of a dimer complex. 4) In the case of the complex containing two inosines instead of guanosines in the stem region, only one signal assigned to the I^.C pair was observed by ^1H-NMR.
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