Isolation of the Interferon-Inducible RNA-Dependent Protein KinasePkrPromoter and Identification of a Novel DNA Element within the 5′-Flanking Region of Human and MousePkrGenes

Abstract

The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of thePkrgene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5′-flanking fragments of thePkrgene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed an element (5′GGAAAACGAAACT3′) involved in IFN inducibility that corresponds to the consensus sequence of the IFN-stimulated response element (ISRE). Comparison of the promoter sequence of the humanPkrgene to that of the mouse homolog identified a novel element (5′GGGAAGGCGGAGTCC3′) immediately upstream of the ISRE element which so far is unique to the human and mousePkrgene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of thePkrpromoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the humanPkrpromoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mousePkrgenes. The strict conservation of sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.