Mechanism of interferon action:: Functional characterization of positive and negative regulatory domains that modulate transcriptional activation of the human RNA-dependent protein kinase Pkr promoter

The PKR protein kinase is an important regulator of viral mRNA translation. A similar to 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. Competition and supershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Spl. In addition to the positive regulatory domain containing the KCS and ISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned similar to 400-bp upstream of the KCS and ISRE 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 positive and negative regulatory domains within the Pkr promoter that function through the KCS element to affect basal and IFN-inducible transcription of Pkr. (C) 1999 Academic Press.