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The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.