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Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

A natural amino acid substitution in the human immunodeficiency virus type 1 (HIV-1) transcriptional activator Tat increases its activity and compensates for deleterious mutations elsewhere in the Tat protein. Substitution of asparagine for threonine 23 increases Tat transactivation of the HIV-1 promoter and the binding of Tat to the cellular kinase positive transcription elongation factor b (P-TEFb). Of nine other position 23 mutations tested, only the serine substitution retained wild-type activity. Correspondingly, asparagine is the most frequent amino acid at this position in HIV-1 isolates, followed by threonine and serine. Asparagine is prevalent in Tat proteins of viruses in clades A, C, and D, which are major etiologic agents of AIDS. We suggest that selection for asparagine in position 23 confers an advantage to the virus, since it can compensate for deleterious mutations in Tat. It may also support the replication of otherwise less fit drug-resistant viruses and permit the emergence of virulent strains.

Over a dozen human genes code for proteins that specifically bind to double-stranded RNA. These proteins have been implicated in several important cellular processes such as transcriptional activation, inhibition of translational initiation, RNA editing, mRNA localization, signal transduction, and posttranscriptional gene silencing (PTGS or RNAi). The recent discovery that PTGS or RNAi is a dsRNA-mediated pathway has further added to the study of dsRNA binding proteins. A method that enables the cloning of genes encoding dsRNA binding proteins would greatly facilitate the identification and study of these molecules. Here we describe a method for isolating such genes from an expression library using radiolabeled poly(I):poly(C) as a binding substrate.

Like other HIV-1 (human immunodeficiency virus type 1) proteins, Tat undergoes rapid mutation and occurs in numerous sequence variants in nature. Virus isolated from patients often has defects in Tat that lower its activity. The levels of P-TEFb, an essential cellular cofactor for Tat, are elevated by T-cell activation. To test the hypothesis that stimulation of P-TEFb levels might compensate for attenuation of Tat activity, we generated Tat constructs with a range of transactivation function. Transactivation by the Tat mutants correlated with their ability to bind to P-TEFb in vitro. Treatment of U937 cells with the phorbol ester PMA (phorbol myristate acetate) induced P-TEFb and stimulated Tat transactivation for alleles with basal transcription activity above a threshold (>5% compared to wild-type). Highly active alleles (>66% of wild-type) were stimulated to a lesser extent than those with activity in the intermediate range. Thus, attenuation of Tat function, in concert with low levels of P-TEFb activity, could serve to keep the virus in a latent state in quiescent cells yet permit viral replication after cell activation.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

RNA helicase A (RHA) is a multifunctional protein involved in various nuclear processes such as transcription and RNA export. It is believed that the interacting factors play important roles in determining the functional specificity of RHA. Here we show that RHA directly interacts with double-stranded (ds) nucleic acids (NAs) and assembles complexes with topoisomerase IIalpha. First, electrophoresis mobility shift assays demonstrate that RHA interacts with dsDNAs of different lengths ranging from 15 to 104 bp. Secondly, the binding of RHA to closed circular dsDNA stimulates the relaxation reaction catalyzed by either calf thymus topoisomerase I or HeLa topoisomerase IIalpha. Thirdly, immunoprecipitation, coupled with western blot analysis using anti-RHA and anti-topoisomerase IIalpha antibodies, shows that RHA and topoisomerase IIalpha assemble a complex in the presence of as yet unknown RNA molecules and additional protein factors such as Ubc9. Our observation suggests physical and functional interaction between RHA and topoisomerase IIalpha, which, perhaps, play important roles in regulating chromatin structure. The putative role of RHA-topoisomerase IIalpha complex in RNA polymerase II-mediated transcription is discussed.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

The interferon-inducible, double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays key roles in regulation of cell growth and differentiation, and has been postulated as a tumor suppressor. Downstream effectors of PKR include the translation initiation factor, eIF2alpha, and the transcription factor, NF-kappaB. We found elevated levels of PKR protein, dsRNA-dependent PKR autophosphorylation activity, and phosphorylated eIF2alpha in melanoma cells compared to nontransformed melanocytes in culture. Treatment with interferon-alpha2b further induced PKR expression and activity. Immunohistochemical analysis of primary melanomas demonstrated minimal PKR immunoreactivity, but melanoma lymph node metastases expressed a high level of PKR protein. Furthermore, analysis of colon cancer specimens revealed that transformation from normal mucosa to adenomas and carcinomas was coincident with an increase in PKR expression. These data do not support the concept of PKR as a classic tumor suppressor but instead suggest that PKR upregulation occurs at defined steps in cancer progression, probably as a cellular response to neoplasia.

Cytoskeletal proteins are associated with actin in the microfilaments and have a major role in microfilament assembly and function. The expression of some of these proteins has been implicated in cell growth and transformation. Specifically, the 3'-untranslated regions (3'-UTRs) of tropomyosin, troponin and cardiac actin can induce muscle cell differentiation and appear to function as tumor suppressors. These RNA sequences are predicted to fold to form secondary structures with extended stretches of duplex. We show that the 3'-UTRs of the cytoskeletal mRNAs interact with the RNA-binding domain of the RNA-activated protein kinase PKR. Correspondingly, these RNAs activate PKR in vitro and inhibit globin translation in the rabbit reticulocyte lysate translation system. These data are consistent with a mechanism whereby PKR mediates the differentiation- and tumor-related actions of the cytoskeletal 3'-UTR sequences.

Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.

PKR is a double-stranded (ds) RNA activated protein kinase whose expression is induced by interferon. Activated PKR phosphorylates its cellular substrate, eIF2, an essential initiation factor of translation. Prior evidence from a murine model system suggested that PKR may act as a tumor suppressor, but the evidence from human tumors is equivocal. To study PKR function in human breast cancer, PKR activity was measured in mammary carcinoma cell lines and nontransformed mammary epithelial cell lines. If PKR functioned as a tumor suppressor in this system, its activity would be higher in nontransformed cells than in carcinoma cells. On the contrary, PKR autophosphorylation and the phosphorylation of its substrate, the alpha-subunit of eIF2, is 7 - 40-fold higher in lysates prepared from breast carcinoma cell lines than in those from nontransformed epithelial cell lines. Correspondingly, a larger proportion of eIF2alpha is present in a phosphorylated state in carcinoma cell lines than in nontransformed cell lines. Protein synthesis is not inhibited by the high eIF2alpha phosphorylation in carcinoma cells, probably because they contain higher levels of eIF2B, the initiation factor that is inhibited by eIF2alpha phosphorylation. The dramatically lower PKR activity in nontransformed cell lines is partially due to lower PKR protein levels (2 - 4-fold) as well as to the presence of a PKR inhibitor. The nontransformed cells contain P58, a known cellular inhibitor of PKR that physically interacts with PKR and may be responsible for the low PKR activity in these cells. Taken together, these observations call into question the role of PKR as a tumor suppressor and suggest a positive regulatory role of PKR in growth control of breast cancer cells.

Highly structured and double-stranded (ds) RNAs are adaptable and potent biochemical entities. They interact with dsRNA-binding proteins (RBPs), the great majority of which contain a sequence called the dsRNA-binding motif (dsRBM). This approximately 70-amino-acid sequence motif forms a tertiary structure that interacts with dsRNA, with partially duplexed RNA and, in some cases, with RNA-DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or more dsRBMs. The motif also participates in complex formation through protein-protein interactions.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes., (Copyright 1999 Academic Press.)

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.

PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR., (Copyright 1998 Academic Press.)

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425-437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107's ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107's ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732-3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

The Tat protein is a potent activator of human immunodeficiency virus type 1 transcription. Tat has been shown to act by increasing both transcription initiation and elongation, but a detailed understanding of its interaction with the transcriptional machinery is lacking. With the aim of isolating cellular proteins that interact with Tat and play a role in transactivation, we have reexamined its function in a cell-free transcription assay. Monitoring the appearance of transactivation after addition of purified Tat at intervals to the reaction mix revealed a lag of approximately 10 min before Tat is able to effect transactivation. Incubation of Tat in nuclear or cytoplasmic extracts of human cells was sufficient to eliminate the lag, but nuclear extract from a rodent cell line was inactive. The accelerating effect of the human cell extract could be abrogated by dilution, heat inactivation, or chromatographic depletion. We infer that Tat is potentiated for transactivation through interaction with a protein factor(s) that is specific to human cells.

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Transcription of the human immunodeficiency virus type 1 (HIV-1) genome takes place after integration of the provirus into human chromosomal DNA. HIV transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. To investigate the activity of the integrated HIV promoter, we exploited the ability of recombinant adeno-associated virus (AAV-2) to transfer and stably integrate genes into the human genome at random or site-specifically. Chimeric AAV vectors were constructed containing an HIV-CAT reporter cassette; some vectors also contained the neomycin resistance gene to facilitate the isolation of positive clones. HeLa cells were infected with recombinant AAV, in some instances together with wild-type virus as a source of AAV rep function. We isolated 25 clones of G418-resistant cells which carried the integrated HIV-CAT cassette, generally occupying unique sites that did not correspond to the AAV-specific region of chromosome 19. The HIV promoter was transcriptionally active in most of the clones. Basal promoter activity varied substantially among the clones, and its responsivity to the HIV transactivator Tat was also variable. The integrated HIV promoter was transactivated to comparable degrees by the one-exon form and two-exon form of Tat. These findings provide evidence that the transcriptional activity of the HIV promoter can be greatly influenced by the site of proviral insertion.

The proliferating cell nuclear antigen (PCNA), a crucial component of eukaryotic cell cycle and DNA replication complexes, is induced by the adenovirus E1A 243R oncoprotein through a cis-acting element termed the PERE (PCNA-E1A responsive element). The PERE contains a sequence homologous to an activating transcription factor (ATF) motif, and ATF-1 is a major component of PERE-protein complexes. We have identified a second PERE-binding protein, the cAMP response element binding protein (CREB) transcription factor, which forms heterodimers with ATF-1 at this site. CREB, but not ATF-1, is able to mediate transactivation of a minimal PCNA-chloramphenicol acetyltransferase reporter by E1A 243R. Further analysis revealed that the transcriptional coactivator, the CREB-binding protein (CBP), associates with PERE-related complexes, and that CBP is able to mediate a strong transactivation response to E1A 243R at the PCNA promoter. Experiments conducted with mutants in the E1A or CREB components support a model whereby E1A 243R transactivates the PCNA promoter via a CBP-CREB-PERE pathway. These findings delineate a paradigm by which E1A 243R can target and transactivate specific DNA promoter sequences.

The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis. It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA. The best-studied inhibitor of PKR activation is adenovirus VA RNA1. Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described. A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed. A scheme to analyze the RNA product with specific nucleases is discussed. In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation. An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed.

A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity.

We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

The small (160 nt) adenovirus RNA, VA RNAI, antagonizes the activation of the cellular protein kinase PKR (also known as DAI), a key regulator of gene expression. VA RNA consists of two stems separated by a complex region, the central domain, that is essential for its function. A notable feature of the central domain is a pair of tetranucleotides, GGGU and ACCC, which are mutually complementary and phylogenetically conserved. To investigate their role in the structure and function of VA RNA, we generated three sets of mutations designed to disrupt the putative stem and to restore it with different nucleotides. Substitutions in either of the tetranucleotides abrogated VA RNA function in two independent PKR-based assays, demonstrating the importance of these sequences in vivo. Compensating mutants restored function, indicating that the two tetranucleotides pair in the cell, but all of the compensating mutants were less active than wild-type VA RNA. The effects of the mutations on RNA structure were probed by nuclease sensitivity analysis. Pronounced changes in two loops in the central domain correlated closely with the formation and disruption of the stem, suggesting that the tetranucleotide stem defines a critical element in the structure of the central domain through tertiary interactions with the two loops. A model for the central domain is presented that accommodates these findings and also accounts for the known sites of PKR interaction.

The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.

To explore the structure and function of a small regulatory RNA, we examined the virus-associated (VA) RNA species of all 47 known human adenovirus serotypes and of one simian virus, SA7. The VA RNA gene regions of 43 human adenoviruses were amplified and sequenced, and the structures of 10 representative VA RNAs were probed by nuclease sensitivity analysis. Most human viruses have two VA RNA species, VA RNA, and VA RNAII, but nine viruses (19%) have a single VA RNA gene. Sequence alignments classified the RNAs into eight families, corresponding broadly to the known virus groups, and three superfamilies. One superfamily contains the single VA RNAs of groups A and F and the VA RNAI species of group C; the second contains the VA RNAI species of groups B1, D, and E and the unclassified viruses (adenovirus types 42 to 47), as well as the single VA RNAs of group B2; and the third contains all VA RNAII species. Fourteen regions of homology occur throughout the molecule. The longest of these correspond to transcription signals; most of the others participate in RNA secondary structure. The previously identified tetranucleotide pair, GGGU:ACCC, is nearly invariant, diverging slightly (to GGGU:ACCU) only in the two group F viruses and forming a stem in the central domain that is critical for VA RNA structure and function. Secondary structure models which accommodate the nuclease sensitivity data and sequence variations within each family were generated. The major structural features-the terminal stem, apical stem-loop, and central domain-are conserved in all VA RNAs, but differences exist in the apical stem and central domains, especially of the VA RNAII species. Sequence analysis suggests that an ancestral VA RNA gene underwent duplication during the evolution of viruses containing two VA RNA genes. Although the VA RNAII gene seems to have been lost or inactivated by secondary deletion events in some viruses, the high degree of homology among the VA RNAII species implies that this RNA may play an undiscovered role in virus survival. We speculate that the VA RNA genes originated from cellular sequences containing multiple tRNA genes.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

A model is presented for the regulation of the double-stranded RNA (dsRNA)-activated mammalian protein kinase PKR, which is involved in protein synthesis inhibition and the antiviral response in cells. A series of previous findings abut PKROs behavior are reviewed, including its effects on translation; the activation of its protein kinase activity; binding sites for PKR on RNA; PKROs protein domains, which include two double-stranded RNA binding motifs (dsRBMs); and the likelihood of PKR dimer formation. The model which emerges to account for many of these observations includes the suggestion that PKR dimers form which are stabilized and rearranged upon binding to dsRNA regions 60 bp or longer. The hypothesis includes protein conformational changes within each member of a PKR dimer bound to dsRNA which re-position an inhibitory polypeptide domain and thus allow kinase activation. Also considered are ways in which PKR interacts with imperfectly duplexed, highly structured RNA molecules.