RESUMO
The sera of six patients with autoimmune disease, predominantly myositis with pulmonary fibrosis, contain antibodies of the PL-12 specificity. These autoantibodies react with both protein and RNA components of human cells. The protein has a subunit molecular mass of 110 kD, and the RNA comprises a group of bands in the tRNA size class. Aminoacylation experiments identify the antigens as alanyl-tRNA synthetase and its corresponding tRNAs, tRNAAla. Anti-tRNA antibody can be absorbed out without depleting antisynthetase activity, showing that the antigens are recognized independently by separable antibodies that coexist in these sera. The concurrence of separate antibodies to the two components suggests that the autoimmune response may be mounted against the charging enzyme-tRNA complex. However, the antisynthetase antibody fails to coprecipitate tRNA with the enzyme, suggesting that the antibody reacts with its target only when it is not complexed with tRNA.
Assuntos
Alanina-tRNA Ligase/imunologia , Aminoacil-tRNA Sintetases/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Miosite/imunologia , RNA de Transferência/imunologia , Adulto , Alanina , Alanina-tRNA Ligase/antagonistas & inibidores , Especificidade de Anticorpos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
An autoantibody known as PL-7 was found in the serum of four patients with myositis and one with a systemic lupus erythematosus-like syndrome. The PL-7 antigen is an 80,000 dalton polypeptide that coprecipitates with transfer RNA. In aminoacylation reactions, PL-7 IgG inhibited the charging of tRNA with threonine but had little or no effect on charging with other amino acids. Experimental antibodies raised against purified threonyl-tRNA synthetase recognized the same 80,000 dalton polypeptide, but tRNA was not coprecipitated. We conclude that PL-7 antibody is directed at threonyl-tRNA synthetase, and that different antigenic sites are recognized by the human and experimental autoantibodies. Our findings emphasize the link between myositis and autoimmunity to tRNA-related structures.
Assuntos
Aminoacil-tRNA Sintetases/imunologia , Autoanticorpos/análise , Miosite/imunologia , Treonina-tRNA Ligase/imunologia , Adulto , Animais , Especificidade de Anticorpos , Autoanticorpos/fisiologia , Autoantígenos/análise , Ligação Competitiva , Epitopos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Precipitina , RNA/análise , Coelhos , Treonina-tRNA Ligase/isolamento & purificaçãoRESUMO
Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.
Assuntos
Anticódon/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Miosite/imunologia , RNA de Transferência de Alanina/imunologia , RNA de Transferência Aminoácido-Específico/imunologia , RNA de Transferência/imunologia , Alanina-tRNA Ligase/imunologia , Epitopos , Conformação de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Rabbit aortic medial cells were grown on purified elastin membranes, which were then subjected to repeated elongation and relaxation or to agitation without stretching. Cells remained attached to the membranes, and cyclic stretching resulted in a two- to fourfold increase in rates of collagen, hyaluronate, and chondroitin 6-sulfate synthesis over those in agitated or stationary preparations. Synthesis of types I and III collagen was increased to the same degree. Stretching did not increase rates of chondroitin 4-sulfate or dermatan sulfate synthesis. Differences were not attributable to differences in cell number, for DNA synthetic rates were not increased by stretching. The model system devised to demonstrate these effects provides a means for relating various modes of mechanical stimulation to cell metabolism.
Assuntos
Aorta/metabolismo , Colágeno/biossíntese , Glicosaminoglicanos/biossíntese , Contração Muscular , Proteínas Musculares/biossíntese , Músculo Liso/metabolismo , Animais , Aorta/citologia , Bovinos , Células Cultivadas , DNA/biossíntese , Elastina , PeriodicidadeRESUMO
X-ray diffraction shows that chondroitin 6-sulfate, and some further rulfated derivatives, can occur in two ordered structures in stretched films. Both structures contain single helices with similar projected disaccharide lengths (9.6 and 9.8 angstroms) but with very different turn angles between successive disaccharides (120 and 45 degrees). In contrast, coaxial double helices of hyaluronates and t-carrageenates have shorter projected disaccharide lengths (8.5 and 8.9 angstroms).
Assuntos
Condroitina , Glicosaminoglicanos , Carragenina , Ácido Hialurônico , Modelos Estruturais , Conformação Molecular , Difração de Raios XRESUMO
The Cold Spring Harbor Symposium on The Ribosome was held on 31 May - 5 June in Cold Spring Harbor, NY. USA.
Assuntos
Genoma , Ribossomos/genética , Ribossomos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , RNA/genética , RNA/metabolismo , RNA Ribossômico/química , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/ultraestruturaRESUMO
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.
Assuntos
Motivos de Aminoácidos , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Ligação a RNA/química , Homologia de Sequência de AminoácidosRESUMO
The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.
Assuntos
Proteínas Quinases/isolamento & purificação , Sítios de Ligação , Ligação Competitiva , Ativação Enzimática , Humanos , Cinética , Peso Molecular , Fosforilação , Conformação Proteica , Proteínas Quinases/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , eIF-2 QuinaseRESUMO
In transient expression assays, the adenovirus E1B 19-kilodalton (19K) tumor antigen increases expression from viral promoters and the promoter for the cellular 70-kilodalton heat shock protein (hsp70). To study the mechanism of this effect, we constructed HeLa cell lines that contain stably integrated copies of the 19K gene. Compared with a 19K- control cell line, 19K+ cells produced a significantly higher level of expression from every promoter introduced into the cells by transfection. The 19K protein also increased expression of an RNA polymerase III-transcribed gene but did not affect the level of expression of the endogenous hsp70 gene. The rate of transcription from transfected promoters, as measured by a nuclear run-on assay, was higher in the 19K+ cells than in the 19K- control cells. Furthermore, the level of plasmid DNA remained higher in the 19K+ cell line, suggesting that the 19K protein stabilizes transfected plasmid DNA. The elevated DNA levels seemed to account in full for the increased transcription. The role of the 19K protein in increasing gene expression during viral infection was found to be due to a replication-dependent increase in viral DNA levels. Thus, the 19K protein activates transcription indirectly by producing a higher level of viral or plasmid DNA. The DNA stabilization function of the 19K protein is probably related to the protective role of the 19K protein during viral infection and represents the first example of a viral oncogene product that modulates gene expression by regulating viral and plasmid DNA levels.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Viral , DNA de Neoplasias/biossíntese , Expressão Gênica , Genes , Proteínas de Choque Térmico/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas Precoces de Adenovirus , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Células HeLa/metabolismo , Humanos , Peso Molecular , Mutação , Proteínas Oncogênicas Virais/genética , Plasmídeos , Regiões Promotoras Genéticas , TransfecçãoRESUMO
Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
Assuntos
Biossíntese de Proteínas , RNA Polimerase III/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Sequência de Bases , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Produtos do Gene tat/biossíntese , Produtos do Gene tat/genética , Genes Reporter , HIV-1/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Polirribossomos/metabolismo , Capuzes de RNA/análise , RNA Mensageiro/análise , RNA Nuclear Pequeno/genética , Proteínas Recombinantes de Fusão/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.
Assuntos
Nucleotídeos de Adenina/metabolismo , Antígenos/metabolismo , Oligonucleotídeos/metabolismo , Oligorribonucleotídeos/metabolismo , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Adenovírus Humanos/genética , Autoantígenos , Células HeLa/metabolismo , Humanos , Cinética , Peso Molecular , RNA Viral/genética , Antígeno SS-BRESUMO
Amino acid analogs, like other effectors of the stress response, induce in mammalian cells the same gene products that are induced upon heat shock; incorporation of the analog into protein is required for induction. We show here that induction by analogs involves controls operating at the levels of both transcription and translation. The electrophoretic patterns of newly made mRNAs simplify with time such that the putative stress protein mRNAs are the only species transported from the nucleus. Concomitantly, the patterns of protein synthesis simplify such that the stress proteins become nearly exclusive polypeptide products. Although the normal mRNAs are either not used or used with greatly reduced efficiency, they are not degraded and retain translatability when transferred to cell-free systems. Soon after the stress response has been induced, there follows a defect in the initiation of polypeptide chains, as evidenced by examination of polysome profiles. Upon prolonged exposure, polysomes are recovered, and although they give rise to stress proteins almost exclusively, the normal mRNAs are still present in these structures. Thus, in addition to the initiation defect, a lesion in elongation may also be involved. The extreme sensitivity of protein synthesis to the inhibition of RNA synthesis, together with the parallel simplifications in the patterns of newly made mRNAs and polypeptides, may imply that only newly made mRNAs are efficiently translated in analog-treated cells.
Assuntos
Ácido Azetidinocarboxílico/toxicidade , Azetinas/toxicidade , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Cicloeximida/toxicidade , Dactinomicina/toxicidade , Desoxiadenosinas/toxicidade , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Cinética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , RNA Mensageiro/genéticaRESUMO
The adenovirus E1A oncoproteins interfere with the normal regulation of cellular proliferation through interactions with cell cycle regulatory proteins. In view of the essential role of proliferating-cell nuclear antigen (PCNA) in DNA replication, we performed a mutational analysis of the minimal human PCNA promoter (nucleotides -87 to +62) to define sequence elements which mediate transactivation by the 243-residue E1A protein (E1A 243R). Linker-scanning and site-directed mutants were examined for basal and E1A-induced expression of chloramphenicol acetyltransferase (CAT) from PCNA promoter-CAT reporter constructs transiently expressed in HeLa cells. The results define the cis-acting element required for induction of PCNA by E1A 243R as a region between -59 and -45 relative to the transcription initiation site. This PCNA E1A-responsive element (PERE), which is protected from DNase I digestion by nuclear extracts from 293 cells, includes the sequence AGCGTGG immediately upstream of the ATF binding site previously shown to be important for activation of PCNA by E1A 243R (G. F. Morris and M. B. Mathews, J. Virol. 65:6397-6406, 1991). Mutation of either the upstream component or the ATF site within the PERE diminishes basal promoter activity and abrogates transactivation by E1A 243R. This novel cis-acting element is also essential for both basal and E1A-induced expression in the context of the full-length PCNA promoter.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Antígenos de Neoplasias/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ativação Transcricional , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Análise Mutacional de DNA , Variação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação , Alinhamento de Sequência , Transcrição GênicaRESUMO
Previous analyses defined a proliferating cell nuclear antigen (PCNA) E1A-responsive element (PERE) in the PCNA promoter that is essential for transactivation by the 243-residue product of the adenovirus type 2 E1A 12S mRNA (E1A 243R). In this report, we show that the PERE activates a heterologous basal promoter and confers susceptibility to transactivation by E1A 243R, indicating that the PERE is both necessary and sufficient for the response of the PCNA promoter to this oncoprotein. Insertion of linker sequences between the PERE and the site of transcription initiation in the PCNA promoter severely impairs the promoter's response to E1A 243R transactivation. GAL4 sites can replace the function of the PERE in the E1A 243R response of the PCNA basal promoter if transcriptional activators of suitable strength are supplied as GAL4 fusion proteins. Weak transcriptional activators render the PCNA basal promoter subject to transactivation by E1A 243R but do not endow the adenovirus E1B basal promoter with a similar response. Strong transcriptional activators do not support transactivation by E1A 243R, however; instead, E1A reduces the ability of the strong activators to activate both the PCNA and E1B basal promoters. Although other mechanistic differences might determine the response, the data imply a relationship between the activation strength of promoter-proximal effectors and the response of the PCNA basal promoter to E1A 243R. These experiments indicate that the PERE can function autonomously in mediating transactivation by E1A 243R and that the PCNA basal promoter is configured in a manner that permits modulation by E1A 243R of transcriptional activation by promoter-proximal effectors.
Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação , Proteínas Recombinantes de Fusão/genética , Transativadores/genética , Fator de Transcrição TFIID , Fatores de Transcrição/genética , TransfecçãoRESUMO
The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.
Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Sítios de Ligação , Sequência Consenso , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA de Cadeia Dupla/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Viral/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , eIF-2 QuinaseRESUMO
The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.
Assuntos
Adenovírus Humanos/genética , Genes Virais , Genes , Proteínas Oncogênicas Virais/genética , Proteínas Precoces de Adenovirus , Animais , Sequência de Bases , Linhagem Celular , Deleção Cromossômica , Enzimas de Restrição do DNA , Células HeLa/metabolismo , Humanos , Rim , Mutação , Plasmídeos , RatosRESUMO
HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.
Assuntos
Antígenos/isolamento & purificação , Autoantígenos/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Núcleo Celular/análise , Células HeLa/análise , Humanos , Fosfoproteínas/isolamento & purificação , Fosforilação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.
Assuntos
Ciclo Celular , Genes Virais , Proteínas Oncogênicas Virais/genética , Proteínas Precoces de Adenovirus , Animais , Bioensaio , Células Cultivadas , Replicação do DNA , DNA Viral/biossíntese , Regulação da Expressão Gênica , Células HeLa , Mitose , Mutação , Nucleoproteínas/genética , Antígeno Nuclear de Célula em Proliferação , Ratos , Timidilato Sintase/genética , Replicação ViralRESUMO
The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.
Assuntos
Proteínas Quinases/metabolismo , RNA de Cadeia Dupla/metabolismo , Sítios de Ligação , Ativação Enzimática , Inibidores de Proteínas Quinases , RNA de Cadeia Dupla/síntese química , eIF-2 QuinaseRESUMO
The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.