Alterations in the p′R promoter of coliphage λ modify both its activity and interaction with the integration host factor (IHF)

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Abstract
A limited number of deletion/insertions and a point mutation in the −35 region of the p′R promoter of phage λ were examined and found to influence both transcription and its repression by the integration host factor (IHF). Positive effects on transcription (in the absence of IHF) are small (up to 1.4-fold) and are caused by a deletion-substitution upstream of the −35/ihf site. Up to three base changes in the −35 promoter element seem to be tolerated, with only a small negative effect on transcription. In some cases, effective transcription requires supercoiling of such mutant template. Since an ihf sequence overlaps the −35 region of p′R, IHF represses transcription. Repression is correlated with IHF binding and consequent DNA bending, as assessed by gel retardation experiments. Nine p′R mutants were tested for their IHF binding and repression; the results confirm the consensus sequence, 5′-W2WWWWN7WWWWCARNWN2TTR derived from the hydroxyl radical footprinting, where the bold letters indicate the IHF-protected bases and W is A or T, R is A or G and N represents A, T, G or C. Perhaps surprisingly, some mutations just upstream or downstream of this ihf sequence also affect IHF binding, as observed not only for the p′R/ihf but also for the att H′ site of λ. Supercoiling in some cases permits the IHF-mediated repression to be overcome, probably by increasing the RNA polymerase binding and/or decreasing the interaction with IHF. All our data are consistent with a model which assumes that IHF initially binds to one or two ihf contact points depending on preexisting DNA topology, bends DNA, and completes the remaining contacts while finally adjusting the DNA conformation to establish the best fit within the minor groove of the double helix. Effective IHF repression of transcription would thus depend on several factors, including: (1) the sequence, and (2) the initial conformation of the ihf site, together with (3) the capacity of IHF to compete with RNA polymerase for the overlapping binding sites.