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由超螺旋产生的变化尚未完全理解。在本研究项目中，我们首先测量了大肠杆菌lac启动子及其突变体启动子在超螺旋密度从0到-0.09的不同值下的体外转录活性。结果发现，转录活性的增加与负的超螺旋密度的增加是较大的，这样的启动子，这是弱的比那些强时，超螺旋是缺席。此外，当在弱启动子的-59位插入胞嘧啶-鸟嘌呤交替序列（CG）n时，上述新的转录本的出现与弱启动子中的转录本的出现有关。 关于我们 作为理解超螺旋在DNA三维结构方面对转录产生这些影响的机制的第一步，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10个或更多个启动子。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，并且当我们将+1碱基对置于原点时，弯曲方向为y轴的负方向，-35区域沿z轴的正方向沿着，并且根据Dickerson的建议，沿x轴和y轴。这一观察结果非常有趣，因为有人提出，通过结合RNA聚合酶在-35至-10区域产生的应力诱导下游区域的DNA解链。有必要进一步研究弯曲的程度以及详细的取向与强度之间的关系，以及对启动子超螺旋的响应。这项工作是与R. D.威尔斯博士和Y.西村合作进行的。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