Transcription Start under the Stress of Supercoiling and Template DNA Structure

在原核生物的转录启动反应中，RNA聚合酶识别所谓的-35和-10共有序列。决定每个启动子强度的分子机制以及超螺旋产生的变化还没有完全弄清楚。在本研究项目中，我们首先测定了大肠杆菌lac启动子及其突变启动子在不同超螺旋密度下的体外转录活性，范围从0到-0.09。结果发现，这些启动子的转录活性随负超螺旋密度的增加而增加，且在无超螺旋时弱于强启动子。此外，当引入负超螺旋时，对于弱启动子，可能开始于真实转录本上游的新转录本出现。此外，当在-59位插入胞嘧啶-鸟嘌呤交替序列(CG)n时，上述新转录本的出现在r…中对超级线圈的更多反应变成了相反的：它在低负超级线圈密度下观察到，并在高负超级线圈密度下消失。作为从DNA的三维结构理解超级线圈对转录产生这些影响的机制的第一步，我们使用了一个可以计算DNA螺旋轨迹的程序，并将其应用于10个或更多的启动子。结果表明，对于大多数启动子来说，DNA螺旋轴在-35和1之间的区域弯曲，当我们按照Dickerson的建议将1碱基对放置在原点时，DNA螺旋轴弯曲到y轴的负方向，-35区域沿着z轴放置在正向，x轴和y轴放置在x轴和y轴之间。这一观察非常有趣，因为它暗示，由RNA聚合酶结合在-35至-10区域产生的压力会导致下游区域的DNA融化。有必要进一步研究促进剂的弯曲程度和详细取向与强度之间的关系以及对超卷曲的响应。这项工作是与R.D.Wells博士和Y.Nishimura博士合作进行的。较少

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