Characterization of the integration host factor binding site in the ilvPG1 promoter region of the ilvGMEDA operon of Escherichia coli.
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.
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
J W
JW
Winkelman
G W
GW
Hatfield
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D034982:Integration Host Factors; D007532:Isoleucine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D011401:Promoter Regions, Genetic; D013463:Sulfuric Acid Esters; D014633:Valine / Q000096:biosynthesis*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D013463:Sulfuric Acid Esters; D007532:Isoleucine; D014633:Valine; C007482:dimethyl sulfate