Chan P T, Lebowitz J
Department of Microbiology, University of Alabama, Birmingham 35294.
J Biol Chem. 1990 Mar 5;265(7):4091-7.
Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.
当n为4或更大时，同聚dAn.dTn序列具有特殊性质，可导致双链稳定性增加和DNA弯曲。lacUV5启动子用于研究将-10 TATAAT共有序列改变为TAAAAT序列的功能后果。下划线处位点的颠换突变通过定点诱变完成，使用翻译终止作为选择程序。对于游离DNA，dA4.dT4片段5'和3'连接区域的结构差异可通过DNase I消化轻松检测到。然而，大肠杆菌RNA聚合酶的位点结合似乎未因TAAAAT序列而改变，因为lacUV5和突变型启动子获得了相同的DNase I足迹。在不同离子强度下的结合竞争研究表明，与lacUV5启动子相比，突变型启动子开放复合物的形成显著减少。当用肝素或肝素与增加的KCl浓度的组合进行挑战时，突变型启动子开放复合物也比相应的lacUV5启动子开放复合物解离得更快且程度更大。因此，突变型启动子开放复合物似乎比lacUV5启动子开放复合物更不稳定。
