Kuhnke G, Fritz H J, Ehring R
EMBO J. 1987 Feb;6(2):507-13. doi: 10.1002/j.1460-2075.1987.tb04782.x.
Two mutations are described, each of which renders the Pribnow box sequence of one of the two overlapping promoters of the Escherichia coli galactose operon identical to the consensus sequence TATAAT. Both double exchanges were specifically introduced into the original context by oligonucleotide-directed mutation construction. Each of the mutant promoters exhibits a greatly enhanced capacity to form stable complexes with RNA polymerase, as judged by nuclease protection experiments and by assaying shifts of electrophoretic mobility. On the other hand, the effect of the same mutations on the rates of transcription from the two gal promoters is strikingly different. Unexpectedly, when complexed with RNA polymerase, DNA fragments carrying one of the two double exchanges were found to differ from each other as well as from the corresponding wild-type fragment with respect to their electrophoretic mobilities. These observations are indicative of different three-dimensional structures of these complexes which may reflect different forms of DNA bending induced in these otherwise identical fragments by complex formation with RNA polymerase.
本文描述了两种突变，每种突变都使大肠杆菌半乳糖操纵子两个重叠启动子之一的普里布诺框序列与共有序列TATAAT相同。通过寡核苷酸定向突变构建，这两种双交换都被特异性地引入到原始背景中。通过核酸酶保护实验和检测电泳迁移率的变化判断，每个突变启动子与RNA聚合酶形成稳定复合物的能力都大大增强。另一方面，相同突变对两个半乳糖启动子转录速率的影响却截然不同。出乎意料的是，当与RNA聚合酶复合时，携带两种双交换之一的DNA片段在电泳迁移率方面彼此不同，也与相应的野生型片段不同。这些观察结果表明这些复合物具有不同的三维结构，这可能反映了与RNA聚合酶形成复合物后，这些原本相同的片段中诱导产生的不同形式的DNA弯曲。
