Polypeptide Chain Initiation Research Articles

A Tetrahedral Representation of the Genetic Code Emphasizing Aspects of Symmetry

The genetic code is a mapping of 64 codons to 22 actions, including polypeptide chain initiation, termination, and incorporation of the twenty amino acids. The standard tabular representation is useful for looking up which amino acid is encoded by a particular codon, but says little about functional relationships in the code. The possibility of making sense of the code rather than simply enumerating its codon-to-action pairings therefore is appealing, and many have attempted to find geometric representations of the code that illuminate its functional organization. Here, I show that a regular tetrahedron with each of its four faces divided into sixteen equilateral triangles (for a total of 64 triangular 'cells') is a particularly apt geometry for representing the code. I apply five principles of symmetry and balance in order to assign codons to the triangular cells of the tetrahedral faces. These principles draw on various aspects of the genetic code and the twenty amino acids, making the final construct a positional balance of the amino acids and their functions rather than a re-analysis of them. The potential significance of this exercise, and others like it, is that this way of organizing the biological facts may provide new insights into them.

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Activation of p53 stimulates proteasome‐dependent truncation of eIF4E‐binding protein 1 (4E‐BP1)

The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m(7)GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.

The Bacterial YbaK Protein Is a Cys-tRNAPro andCys-tRNACysDeacylase

Bacterial prolyl-tRNA synthetases and some smaller paralogs, YbaK and ProX, can hydrolyze misacylated Cys-tRNA Pro or Ala-tRNA Pro. To assess the significance of this quality control editing reaction in vivo, we tested Escherichia coli ybaK for its ability to suppress the E. coli thymidylate synthase thyA:146CCA missense mutant strain, which requires Cys-tRNA(Pro) for growth in the absence of thymine. Missense suppression was observed in a ybaK deletion background, suggesting that YbaK functions as a Cys-tRNA Pro deacylase in vivo. In vitro studies with the full set of 20 E. coli aminoacyl-tRNAs revealed that the Haemophilus influenzae and E. coli YbaK proteins are moderately general aminoacyl-tRNA deacylases that preferentially hydrolyze Cys-tRNA Pro and Cys-tRNA Cys and are also weak deacylases that cleave Gly-tRNA, Ala-tRNA, Ser-tRNA, Pro-tRNA, and Met-tRNA. The ProX protein acted as an aminoacyl-tRNA deacylase that cleaves preferentially Ala-tRNA and Gly-tRNA. The potential of H. influenzae YbaK to hydrolyze in vivo correctly charged Cys-tRNA Cys was tested in E. coli strain X2913 (ybaK+). Overexpression of H. influenzae ybaK decreased the in vivo ratio of Cys-tRNA Cys to tRNA Cys from 65 to 35% and reduced the growth rate of strain X2913 by 30% in LB medium. These data suggest that YbaK-mediated hydrolysis of aminoacyl-tRNA can influence cell growth.

Initiation factor modifications in the preapoptotic phase

Recent studies have identified several mechanistic links between the regulation of translation and the process of apoptosis. Rates of protein synthesis are controlled by a wide range of agents that induce cell death, and in many instances, the changes that occur to the translational machinery precede overt apoptosis and loss of cell viability. The two principal ways in which factors required for translational activity are modified prior to and during apoptosis involve (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. In this review, we summarise the principal targets for such regulation, with particular emphasis on polypeptide chain initiation factors eIF2 and eIF4G and the eIF4E-binding proteins. We indicate how the functions of these factors and of other proteins with which they interact may be altered as a result of activation of apoptosis and we discuss the potential significance of such changes for translational control and cell growth regulation.

Translational control in virus-infected cells: models for cellular stress responses

Protein synthesis is regulated at the translational level by a variety of mechanisms in virus-infected cells. Viruses often induce the shut-off of host translation in order to favour the expression of their own genetic information, but cells possess a number of strategies for counteracting such effects of infection. Important regulatory mechanisms include the phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF2, RNA degradation mediated by the 2'5'-oligoadenylate/RNase L system, control of availability of the cap-binding protein eIF4E by its interaction with the 4E-binding proteins and specific proteolytic cleavage of several key initiation factors. Most of these mechanisms are also utilised in uninfected cells in response to a variety of physiological stresses and during the early stages of apoptosis. Thus, mechanisms of translational control during virus infection can provide models for the cellular stress responses observed in a wide range of other circumstances.

Targets and mechanisms for the regulation of translation in malignant transformation.

There is increasing evidence that deregulation of gene expression at the level of mRNA translation can contribute to cell transformation and the malignant phenotype. Two steps in the pathway of polypeptide chain initiation, viz. the assembly of the 43S initiation complex catalysed by polypeptide chain initiation factor eIF2 and the binding of eIF4E to eIF4G during the recruitment of mRNA to the ribosome, have been shown to be likely targets for changes associated with tumorigenesis. The activity of eIF2 is controlled by changes in phosphorylation of the alpha subunit of this factor. The availability of eIF4E for binding to eIF4G is regulated by the phosphorylation of a small family of eIF4E-binding proteins (the 4E-BPs). The activities of the protein kinases and/or phosphatases responsible for the (de)phosphorylation of these substrates may in turn be controlled by cellular and viral oncogenes and tumour-suppressor genes. This review will describe recent aspects of the mechanisms involved, with particular emphasis on the regulation of the eIF2 alpha kinase PKR and the control of 4E-BP phosphorylation by viral gene products, growth-inhibitory cytokines and the tumour-suppressor protein p53.

P53-induced inhibition of protein synthesis is independent of apoptosis.

Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-independent events that can be dissociated from the induction of apoptosis per se.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain.

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30 min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70(S6K)) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4--6 h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4 h of reperfusion only in the cortex. Conversely, p70(S6K) activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2 alpha, eIF2B, eIF4G and p70(S6K) are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70S6K) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the α subunit of eIF2 (eIF2α) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4–6h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4h of reperfusion only in the cortex. Conversely, p70S6K activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2α, eIF2B, eIF4G and p70S6K are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.

Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis.

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.

Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.

Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines.

We have investigated the effect of inducing apoptosis in BJAB and Jurkat cells on the cellular content of several polypeptide chain initiation factors. Serum deprivation results in inhibition of protein synthesis and induction of apoptosis in BJAB cells; at early times, there is selective degradation of polypeptide initiation factor eIF4G but no major losses of other key initiation factors. The disappearance of full length eIF4G is accompanied by the appearance of smaller forms of the protein, including a major product of approximately 76 kDa. Apoptosis induced by cycloheximide results in similar effects. Both total cytoplasmic eIF4G and eIF4G associated with eIF4E are degraded with a half-life of 2-4 h under these conditions. Treatment of serum-starved or cycloheximide-treated cells with Z-VAD.FMK or Z-DEVD.FMK, which inhibit caspases required for apoptosis, protects eIF4G from degradation and blocks the appearance of the ca. 76 kDa product. Exposure of BJAB cells to rapamycin rapidly inhibits protein synthesis but does not lead to acute degradation of eIF4G. In both BJAB and Jurkat cells induction of apoptosis with anti-Fas antibody or etoposide also results in the selective loss of eIF4G, which is inhibitable by Z-VAD.FMK. These data suggest that eIF4G is selectively targeted for cleavage as cells undergo apoptosis and is a substrate for proteases activated during this process.

Isolation and characterization of the cDNA and the gene for eukaryotic translation initiation factor 4G from Drosophila melanogaster.

Recent evidence supports the notion that the eukaryotic polypeptide chain initiation factor (eIF)4G plays a critical bridging role in coordinating other eIF involved in eukaryotic translation initiation. Here we report the isolation and characterization of a 5621-bp cDNA encoding Drosophila eIF4G. The longest ORF predicts a polypeptide of 1666 amino acids with a molecular mass of 183,940 Da and shares 25% amino acid identity with other eIF4G. The 5' untranslated region is 386 nucleotides long and contains seven AUG codons out of frame. The in vitro transcription/translation of the cDNA yielded a major polypeptide, which was specifically immunoprecipitated with an antibody against Drosophila eIF4G. This polypeptide has the same electrophoretic mobility as eIF4G purified from Drosophila melanogaster embryos. A conserved eIF4E-binding motif was found in Drosophila eIF4G. The gene maps at the 102E region of chromosome 4 and spans a genomic region of approximately 16 kb. It was found to contain 15 introns. A single RNA transcript of approximately 5.9 kb was detected by northern blotting of poly(A)-rich RNA prepared from Drosophila adults. The sequence upstream of the transcription initiation site lacks the consensus TATA box, but contains several sequences possibly involved in the regulation of transcription.

An alpha subunit-deficient form of eukaryotic protein synthesis initiation factor eIF-2 from rabbit reticulocyte lysate and its activity in ternary complex formation.

Eukaryotic protein synthesis initiation factor eIF-2 is usually isolated as a heterotrimer (alphabeta gamma). By use of Sephacryl S-300 fractionation an alpha subunit-deficient form of eIF-2 was identified in impure preparations from rabbit reticulocyte lysate and it appeared in these preparations to be still active in formation of the ternary complex (eIF-2.GTP.Met-tRNAi). Subsequently alpha subunit-deficient eIF-2 was further purified and this appeared to have retained ternary complex forming activity. Together with a suggested lack of involvement of the beta subunit this implies that the alpha subunit was not required for activity and the gamma subunit bound both GTP and Met-tRNAi in formation of the ternary complex. The identification and study of alpha subunit-deficient eIF-2 thus elucidated the involvement of the subunits in binding of GTP and Met-tRNAi to produce the ternary complex in polypeptide chain initiation.

Initiation of protein synthesis in eukaryotic cells.

It is becoming increasingly apparent that translational control plays an important role in the regulation of gene expression in eukaryotic cells. Most of the known physiological effects on translation are exerted at the level of polypeptide chain initiation. Research on initiation of translation over the past five years has yielded much new information, which can be divided into three main areas: (a) structure and function of initiation factors (including identification by sequencing studies of consensus domains and motifs) and investigation of protein-protein and protein-RNA interactions during initiation; (b) physiological regulation of initiation factor activities and (c) identification of features in the 5' and 3' untranslated regions of messenger RNA molecules that regulate the selection of these mRNAs for translation. This review aims to assess recent progress in these three areas and to explore their interrelationships.

Double-stranded (ds) RNA Binding and Not Dimerization Correlates with the Activation of the dsRNA-dependent Protein Kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.