Transcription Start under the Stress of Supercoiling and Template DNA Structure

在原核生物的转录起始反应中，已知RNA聚合酶可以识别所谓的-35和-10一致序列。决定每个启动子强度的分子机制以及其超卷曲产生的变化尚未完全了解。在本研究项目中，我们首先测量了大肠杆菌lac启动子及其突变启动子在不同超螺旋密度值（0 ~ -0.09）下的体外转录活性。研究发现，当超螺旋缺失时，弱启动子的转录活性随负超螺旋密度的增加而增加的幅度大于强启动子。此外，当引入负超级螺旋时，弱启动子出现了一个新的转录本，可能是从一个真实转录本的上游开始的。此外，当在-59位插入胞嘧啶-鸟嘌呤交替序列（CG）n时，上述新转录物的出现在r…更多的对超线圈的响应变得相反：在低负超线圈密度下观察到它，在高负超线圈密度下消失。为了从DNA三维结构的角度理解产生这些超螺旋效应的转录机制，我们采用了一个程序，通过该程序可以计算DNA螺旋轨迹，并将其应用于十个或更多启动子。结果表明，对于大多数启动子，DNA螺旋轴在-35和+1位点之间的区域弯曲，当我们将+1碱基对放在原点时，弯曲方向是y轴的负方向，将-35区域沿z轴方向弯曲，根据Dickerson的建议，弯曲方向是x轴和y轴的正方向。这一观察结果非常有趣，因为它与RNA聚合酶结合在-35至-10区域产生的应激诱导下游区域的DNA熔化有关。有必要进一步研究弯曲的程度和详细方向与强度以及启动子对超卷曲的响应之间的关系。这项工作是与博士合作进行的。R.D.Wells和Y.Nishimura。少

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 … More 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