Borowiec J A, Gralla J D
J Mol Biol. 1985 Aug 20;184(4):587-98. doi: 10.1016/0022-2836(85)90305-5.
The rate of open promoter complex formation was measured on lac ps promoter DNA templates differing in negative superhelicity. The templates ranged from fully relaxed to those with numbers of superhelical turns exceeding that of form I plasmid DNA. The observed transcription response had two clearly distinguished phases: an initial rapid rise in rate followed eventually by a precipitous inhibition. The stimulation phase involved a nearly 40-fold increase in rate, which peaks at superhelical densities near that of isolated form I plasmid DNA. The introduction of more negative superhelical turns leads to inhibition. The magnitude of the response and the observation of both increases and decreases suggest that minor differences in superhelicity in vivo could lead to significant increases or decreases in transcription rate. The increase in rate was found to be directly proportional to the free energy of supercoiling; that is, to the square of the superhelical density. We suggest that the energy may be used both for enhanced DNA melting and for changes in DNA structure that alter the helical "face" with which RNA polymerase must interact. A quantitative method is presented that allows simple estimation of differences in the supercoiling response among promoters, both in the presence and in the absence of added factors.
在具有不同负超螺旋度的乳糖 ps 启动子 DNA 模板上测量开放启动子复合物的形成速率。模板范围从完全松弛的到超螺旋匝数超过 I 型质粒 DNA 的那些。观察到的转录反应有两个明显不同的阶段：速率最初迅速上升，最终急剧抑制。刺激阶段涉及速率近 40 倍的增加，在接近分离的 I 型质粒 DNA 的超螺旋密度处达到峰值。引入更多的负超螺旋匝数会导致抑制。反应的幅度以及增加和减少的观察结果表明，体内超螺旋度的微小差异可能导致转录速率显著增加或降低。发现速率的增加与超螺旋的自由能成正比；也就是说，与超螺旋密度的平方成正比。我们认为，该能量可用于增强 DNA 解链以及改变 DNA 结构，从而改变 RNA 聚合酶必须相互作用的螺旋“面”。提出了一种定量方法，该方法允许在存在和不存在添加因子的情况下简单估计启动子之间超螺旋反应的差异。
