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Journal of Virology, December 1998, p. 9934-9939, Vol. 72, No. 12
Interdepartmental Biochemistry and Molecular
Biology Graduate Program,1 and
Department of Molecular, Cellular and Developmental
Biology,2 University of California, Santa
Barbara, Santa Barbara, California 93106
Received 22 June 1998/Accepted 27 August 1998
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.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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

*
Corresponding author. Mailing address: Department of
Molecular, Cellular, and Developmental Biology, University of
California, Santa Barbara, Santa Barbara, CA 93106. Phone: (805)
893-3097. Fax: (805) 893-4724. E-mail:
samuel{at}lifesci.ucsb.edu.
Present address: School of Medicine, University of California, San
Diego, La Jolla, CA 92093-0665.
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