Mechanism of Interferon Action: Functional Characterization of Positive and Negative Regulatory Domains That Modulate Transcriptional Activation of the Human RNA-Dependent Protein KinasePkrPromoter

Abstract

The PKR protein kinase is an important regulator of viral mRNA translation. A ∼50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). ThePkrpromoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bpIFN-stimulatedresponseelement (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5′ flanking region of the humanPkrgene 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 activityin vitrocorrelated with activation of transcriptionin vivoin 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 ∼400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affectedPkrtranscription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within thePkrpromoter that function through the KCS element to affect basalIFN-inducible transcription ofPkr.