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The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form., (Copyright 1999 Academic Press.)

The PKR protein kinase is an important regulator of viral mRNA translation. A approximately 50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). The Pkr promoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bp IFN-stimulated response element (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5' flanking region of the human Pkr gene to define how the novel KCS element acts to affect basal as well as IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) revealed that nuclear proteins bound selectively to the KCS element in a manner that was not dependent upon either IFN treatment or protein binding at the adjacent ISRE element. KCS protein binding activity in vitro correlated with activation of transcription in vivo in transient transfection assays. Competitionsupershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Sp1. In addition to the positive regulatory domain containing the KCSISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned approximately 400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affected Pkr transcription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within the Pkr promoter that function through the KCS element to affect basalIFN-inducible transcription of Pkr., (Copyright 1999 Academic Press.)

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 the Pkr gene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the Pkr gene, 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 human Pkr gene 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 mouse Pkr gene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of the Pkr promoter 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 human Pkr promoter 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 mouse Pkr genes. 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.