Winkelman J W, Hatfield G W
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
J Biol Chem. 1990 Jun 15;265(17):10055-60.
The ilvGMEDA operon of Escherichia coli, which encodes four of the five enzyme activities required for the biosynthesis of isoleucine and valine, is preceded by tandem promoters ilvPG1 and ilvPG2 which are separated by 72 base pairs. While both of these promoters are transcriptionally active in vitro, only the operon proximal promoter, ilvPG2, is transcriptionally active in vivo, and upstream DNA sequences encoding the ilvPG1 promoter region enhance the in vivo transcriptional activity of the ilvPG2 promoter 60-fold. The binding of the integration host factor protein (IHF) to this upstream region (Tsui, P., and Freundlich, M. (1989) J. Mol. Biol. 203, 817-820) has been shown to repress transcription from the ilvPG1 promoter both in vivo and in vitro (Pereira, R. F., Ortuno, M. J., and Lawther, R. P. (1988) Nucleic Acids Res. 16, 5972-5989). Furthermore, E. coli strains deficient for IHF are compromised for isoleucine and valine biosynthesis (Friden, P., Voelkel, K., Sternglantz, R., and Freundlich, M. (1984) J. Mol. Biol. 172, 573-579). Therefore, in order to further understand this repressor/activator role of IHF, we have undertaken a detailed analysis of the interaction of IHF with the DNA sequences in the ilvPG1 promoter region. The results of hydroxyl radical footprinting, dimethyl sulfate protection, and ethylation interference experiments show that IHF binds to a target site that overlaps the ilvPG1 promoter region. The results of these experiments also demonstrate that IHF interacts primarily with the minor groove of the DNA helix and that the IHF target site in the ilvPG1 promoter region shares a high degree of DNA sequence identity with other high affinity IHF target sites involved in DNA replication and site-specific recombination.
大肠杆菌的ilvGMEDA操纵子编码异亮氨酸和缬氨酸生物合成所需的五种酶活性中的四种，其前面有串联启动子ilvPG1和ilvPG2，它们相隔72个碱基对。虽然这两个启动子在体外都具有转录活性，但只有操纵子近端启动子ilvPG2在体内具有转录活性，并且编码ilvPG1启动子区域的上游DNA序列可将ilvPG2启动子的体内转录活性提高60倍。整合宿主因子蛋白（IHF）与该上游区域的结合（Tsui, P.和Freundlich, M.（1989年）《分子生物学杂志》203卷，817 - 820页）已显示在体内和体外均可抑制ilvPG1启动子的转录（Pereira, R. F., Ortuno, M. J.和Lawther, R. P.（1988年）《核酸研究》16卷，5972 - 5989页）。此外，缺乏IHF的大肠杆菌菌株在异亮氨酸和缬氨酸生物合成方面存在缺陷（Friden, P., Voelkel, K., Sternglantz, R.和Freundlich, M.（1984年）《分子生物学杂志》172卷，573 - 579页）。因此，为了进一步了解IHF的这种阻遏/激活作用，我们对IHF与ilvPG1启动子区域的DNA序列之间的相互作用进行了详细分析。羟基自由基足迹法、硫酸二甲酯保护法和乙基化干扰实验的结果表明，IHF与一个与ilvPG1启动子区域重叠的靶位点结合。这些实验的结果还表明，IHF主要与DNA螺旋的小沟相互作用，并且ilvPG1启动子区域中的IHF靶位点与参与DNA复制和位点特异性重组的其他高亲和力IHF靶位点具有高度的DNA序列同一性。
