Abstract
Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.
Highlights
The chromosomal DNA of the bacterium Escherichia coli is highly compacted and negatively supercoiled [1,2,3]
integration host factor (IHF) forms a higher-order protein-DNA complex in UAS1 that facilitates unwinding of the DNA helix in the Ϫ10 hexanucleotide region of the downstream ilvPG promoter, and this binding is accompanied by an increase in the rate of opencomplex formation [7]
We show that the activity of the ilvPG promoter increases with increased negative supercoiling and that this sensitivity to superhelical density is enhanced in the presence of IHF
Summary
The chromosomal DNA of the bacterium Escherichia coli is highly compacted and negatively supercoiled [1,2,3]. IHF forms a higher-order protein-DNA complex in UAS1 that facilitates unwinding of the DNA helix in the Ϫ10 hexanucleotide region of the downstream ilvPG promoter, and this binding is accompanied by an increase in the rate of opencomplex formation [7]. These and other observations (see “Discussion”) coupled with the observations that IHF-mediated activation occurs in the absence of specific protein interactions between IHF and RNA polymerase, and that IHF-mediated activation requires a negatively supercoiled DNA template, support the hypothesis that IHF activates transcription from this promoter by an allosteric DNA mechanism that is influenced by the superhelical state of the DNA template [7]. We further show that IHF and DNA supercoiling influence transcription from this promoter by different mechanisms, and that activation is not the consequence of differential binding of IHF to relaxed and supercoiled DNA templates
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