Mechanism of interferon action: identification of essential positions within the novel 15-base-pair KCS element required for transcriptional activation of the RNA-dependent protein kinase pkr gene.
RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (pkr) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the human pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of the pkr promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5' GGGAAGGCGGAGTCC 3'), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mouse pkr promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5' NNRRRGG(C,A,T)GGRGYYN 3', where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.
Kuhen
K L
KL
Interdepartmental Biochemistry and Molecular Biology Graduate Program, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California 93106, USA.
Vessey
J W
JW
Samuel
C E
CE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics*
D017931:DNA Primers; D016898:Interferon-alpha; D009687:Nuclear Proteins; D015500:Chloramphenicol O-Acetyltransferase; D019892:eIF-2 Kinase