| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
In the transcription start reaction of prokaryotes RNA polymerase is known to recognize the so-called -35 and -10 consensus sequences. Molecular mechanisms which determine the strength of each promoter as well as its change produced by supercoiling have not yet fully understood. In this research project, we first measured in vitro transcription activity of Escherichia coli lac promoter and its mutant promoter at various values of superhelical density ranging from 0 to -0.09. It was found that the increase in transcription activity with the increase in the negative superhelical density was larger for such promoters which were weak than those which were strong when supercoil was absent. Also, a new transcript which presumably started upstream of an authentic transcript appeared for the weak promoters when negative supercoil was introduced.
Further, when a cytosine-guanine alternating sequence, (CG)n, was inserted at -59 Position, the appearance of the above-mentioned novel transcript in r
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esponse to supercoil became reversed: it was observed at low negative supercoil and disappeared at high negative supercoil densities.
As a first step toward understanding the mechanisms which produces these effects of supercoiling on transcription in terms of three-dimensional structure of DNA, we employed a program by which a DNA helix trajectory can be calculated and applied it to ten or more promoters. It was shown that for most promoters the DNA helix axis was bent in the region between the -35 and the +1 sites, and the direction of bending was to the minus of the y-axis when we place the +1 base pair at the origin, the -35 region in the plus direction along the z-axis, and the x- and y-axes according to Dickerson's recommendation. This observation is very interesting in connection with the suggestion that the stress produced in the -35 to -10 region by binding of RNA polymerase induces DNA melting in the downstream region. It is necessary to further investigate the relation between the degree as well as detailed orientation of the bending and the strength as well as the response to supercoiling of promoters. This work was carried out in cooperation with Drs. R.D.Wells and Y.Nishimura. Less
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