| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
The E coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid in an ATP-dependent manner. To further elucidate themolecular mechanism of Rho, we have focused on its structural similarity to FィイD21ィエD2-ATPase and conducted the following two lines of studies.
1)Pre-steadystate kinetics of Rho ATPas reaction : Although rho is a homohexamer, several studies in the literature suggest that the six subunits of the hexamer are not functionally identical. Equilibrium nucleotide binding experiments have shown that there are 3-4 tight and 2-3 weak ATP binding sites on the rho hexamer. To understand the role of the two classes of nucleotide binding sites on the rho hexamer, we have measured the kinetics of ATP hydrolysis and nucleotide dissociation from the rho protein. The results indicate that the rho hexamer has 3 noncatalytic sites that do not participate in the fast ATPase turnover, and thus the weak ATP binding sites on rho must be the catalytic sites.
2)Three-dimentional reconstruction of the Rho hexamer ring from electron microscopic images : We have generated a three -dimensional reconstruction of rho at a 20-A resolution, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring . An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring, which in turn sits on the top of the FィイD21ィエD2-like ATPase core domain.
On the basis of these observations, we propose a model for the Rho function consisting of four elementary steps : a) the binding of the rut site on a nascent transcript to the outside of the ring; b)the passage of the transcript into the central channel of the ring; c)the activation of the coupled ATPase-translocation within the central channel; and d)the linear translocation of rho along the transcript towards the polymerase.
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