| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
We would like to make clear the structure how the bent DNA (phased A-tracts), upstream the promoter of plc gane encoding the phospholpase C in Clostridium perfringens, binds to the RNA polymerase (RNAP)α subunit (315 aa) through its C-terminai domain (α CTD, 79 aa). To determine the structrue, the co-crystallization of the phased A-tracts DNA (33 bp) and RNAP α subunit or α CTD is necessary rpaA gene encoding the RNAP α subunit and the grace encoding the α CTD were cloned into pET16b. Each gene was overexpreessed in E.coli BL21(DE3)pLysS. Then both His, tagged proteins were purified as much as available for crystallization. But the amounts of the purified proteins were only a few mg. They did not reads at 50 mg enough for crystallization. Dr.Joshua Sakon (University of Arkansas, U.S.A.) pointed out that the stability of the complex of the phased A-tracts and the α subunit or the α CTD at 25℃ (a certain low temperature) was very important, and should be examined. Since we have not exami
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ned the stability and the method by which the overproduced proteins were purled in a large scale have been not established yet, the co-crystallization of the phased A-tracts DNA and the RNAP α subunit or the α CTD has not been tied.
In parallel, we tried to establish the system of the reconstitution of the RNAP(α2, β, β', σ) to identify the residues of amino-acids involved in the interaction between the phased A-tracts and the α CTD. The β, β', and σ subunits were overproduced in appropriate E.coli strains, and became inclusion bodies. They were solubilized in 6 M urea or guanidine hydrochloride. These solubilized proteins and the His- tagged α subunit or its N-terminal domain (α NTD, 228 aa) were mixed in the reconstitution of the RNAP. The both reconstituted RNAPs, containing the His-α subunit or the His-α NTD, have slight activities at the same level. This suggested that we night introduce the mutations in the α CTD without any influence on the activity, and that the reconstitution of the RNAP would contribute greatly to the studies on the regulation of transcription through the α CTD. Less
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