RESUMO
Multiple alternative interactions between activators and co-activators stimulate transcription by RNA polymerase II. In the past two years, multiprotein co-activator complexes have been characterized and their subunits defined. TATA-box binding protein associated factor (TAF) subunits of yeast TFIID were found to be generally required for transcription in vivo. Mammalian multisubunit coactivator complexes with homologs of the yeast SRB/Mediator subunits have been characterized. Structures of nuclear receptor-coactivator complexes have been determined.
Assuntos
RNA Polimerase II/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Humanos , Fatores de Transcrição/metabolismoRESUMO
Epstein-Barr virus (EBV) evolved an episomal system for maintaining life-long, latent infection of human B lymphocytes. Circular episomes engineered from EBV components required for this latent form of infection have the capacity to persist in most types of replicating mammalian cells without DNA integration and the pitfalls of insertional mutagenesis. EBV episomes are typically transduced using low-efficiency methods. Here we present a method for efficient delivery of EBV episomes to nuclei of hepatocytes in living mice using a helper-dependent adenoviral vector and Cre-mediated recombination in vivo to generate circular EBV episomes following infection. Cre is transiently expressed from a hepatocyte-specific promoter so that vector generation and transgene expression are tissue specific. We show long-term persistence of the circularized vector DNA and expression of a reporter gene in hepatocytes of immunocompetent mice.
Assuntos
Adenoviridae/genética , Herpesvirus Humano 4/genética , Plasmídeos/administração & dosagem , Transgenes/genética , Animais , Linfócitos B/metabolismo , Células Cultivadas , Genes Reporter , Células HeLa , Hepatócitos/metabolismo , Humanos , Imunocompetência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos/química , Transdução GenéticaRESUMO
The adenovirus EIA and pseudorabies virus immediate early (IE) proteins induce transcription from transfected viral and nonviral genes transcribed by RNA polymerase II (class II genes). These proteins have now been shown also to activate transcription of transfected genes transcribed by RNA polymerase III (class III genes). As previously observed for class II genes, this stimulation of class III gene transcription was much greater for transfected genes than for the major endogenous cellular class III genes. Extracts made from cell lines stably expressing a transfected pseudorabies virus IE gene were 10 to 20 times more active in the in vitro transcription of exogenously added class III genes than extracts of the parental cell line. These results indicate that the E1A and IE proteins stimulate the expression of class III genes by a mechanism similar to the mechanism for stimulation of class II gene transcription by these proteins.
Assuntos
Genes Virais , Transcrição Gênica , Proteínas Virais/genética , Adenoviridae/genética , Animais , Drosophila/genética , Células HeLa , Herpesvirus Suídeo 1/genética , Humanos , RNA de Transferência/genética , Coelhos , RatosRESUMO
Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.
Assuntos
Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismoRESUMO
The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.
Assuntos
Regiões Promotoras Genéticas , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Genes , Genes Virais , Humanos , Plasmídeos , Ligação Proteica , RNA de Transferência de Arginina/genética , Fatores de Transcrição/isolamento & purificação , Transcrição GênicaRESUMO
Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.
Assuntos
Adenovírus Humanos/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Precoces de Adenovirus , Genes , Genes Virais , Células HeLa/metabolismo , Humanos , Proteínas Oncogênicas Virais/genética , Regiões Promotoras GenéticasRESUMO
The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.
Assuntos
Proteínas Oncogênicas Virais/genética , Transcrição Gênica , Proteínas Precoces de Adenovirus , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Células HeLa/metabolismo , Humanos , Proteínas Oncogênicas Virais/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
p53, the protein encoded by one of the most significant human tumor suppressor genes, is a sequence-specific transcriptional activator. When activated by a double-stranded DNA break, p53 function arrests cells in G1 and can induce apoptosis. Transcriptional activation function is critical for p53 tumor suppression, although transcriptional repressing and nontranscriptional functions of p53 may contribute. p53 activation requires that it bind to TFIID through interactions with TATA box-binding protein (TBP)-associated factors and potentially with TBP. Here, we studied the mechanism of p53 activation using in vitro transcription and a sufficiently high p53 concentration to squelch activated transcription. Squelching is thought to result when target molecules that interact with activation domains are titrated by binding to excess activator. Addition of either excess TFIIB or TFIID but not other proteins required for p53-activated transcription reversed squelching by high p53 concentrations, whereas neither stimulated transcription in reactions without excess p53. These results reveal that both TFIIB and TFIID are inhibited by high concentrations of p53 and suggest that p53 activation may work through direct or indirect interactions with both TFIIB and TFIID.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Células HeLa , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Fator de Transcrição TFIIB , Fator de Transcrição TFIIDRESUMO
In mammalian and Drosophila cells, the central RNA polymerase II general transcription factor TFIID is a multisubunit complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) bound to the conserved TBP carboxy-terminal core domain. TBP also associates with alternative TAFs in these cells to form general transcription factors required for initiation by RNA polymerases I and III. Although extracts of human HeLa cells contain little TBP that is not associated with TAFs, free TBP is readily isolated from yeast cell extracts. However, recent studies indicate that yeast TBP can also interact with other yeast polypeptides to form multiprotein complexes. We established stable human HeLa cell lines expressing yeast TBP and several yeast-human TBP hybrids to study TBP-TAF interactions. We found that the yeast TBP core domain assembles with a complete set of human TAFs into a stable TFIID complex that can support activated transcription in vitro. The fact that the yeast TBP core, which differs from human TBP core in approximately 20% of its amino acid residues, has the structural features required to form a stable complex with human TAFs implies that Saccharomyces cerevisiae probably contains TAFs that are structurally and functionally analogous to human TAFs. Surprisingly, the non-conserved amino terminus of yeast TBP inhibited association between the yeast core domain and human TAFs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Fúngicas , Células HeLa , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Complexos Multiproteicos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae , Relação Estrutura-Atividade , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIID , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.
Assuntos
Proteínas E1B de Adenovirus/genética , Proteínas Repressoras/genética , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Polimerase II/genética , Proteínas Recombinantes/genética , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIA , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.
Assuntos
Adenoviridae/genética , Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Transcrição Gênica , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Células HeLa/fisiologia , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Fator de Transcrição Sp1 , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismoRESUMO
TFIIA is a general transcription factor that interacts with the TFIID-promoter complex required for transcription initiation by RNA polymerase II. Two lines of evidence suggest that TFIIA is directly involved in the mechanism by which some activators stimulate transcription. First, binding of TFIIA to a TFIID-promoter complex is a rate-limiting step that is enhanced by transcriptional activators GAL4-AH and Zta. Second, recombinant TFIIA greatly enhances activator-dependent transcription. In this study, we found that the activation domains of Zta and VP16 bind directly to TFIIA. Both Zta and VP16 stimulated rapid assembly of a stable TFIID-TFIIA complex on promoter DNA. Analysis of deletion derivatives of the VP16 activation domain indicated that the ability to bind to TFIIA correlates with the ability to enhance TFIID-TFIIA-promoter ternary complex assembly. Thus, we propose that a class of activators stimulate transcription initiation through direct interactions with both TFIIA and TFIID, which stimulate the assembly of an activated TFIIA-TFIID-promoter complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas Virais , Sequência de Aminoácidos , Pegada de DNA , Proteínas Fúngicas/metabolismo , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Fator de Transcrição TFIIA , Fator de Transcrição TFIIDRESUMO
Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Poliovirus/enzimologia , Fatores de Transcrição/metabolismo , Proteínas Virais , Proteases Virais 3C , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , Cisteína Endopeptidases/genética , DNA , Escherichia coli , Células HeLa , Humanos , Dados de Sequência Molecular , Poliovirus/fisiologia , Biossíntese de Proteínas , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIID , Transcrição GênicaRESUMO
Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , TATA Box , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese , Plasmídeos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIID , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/isolamento & purificaçãoRESUMO
The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.
Assuntos
Transformação Celular Viral , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Poliovirus/genética , RNA Polimerase III/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica , Linhagem Celular , Desoxirribonuclease I , Células HeLa/enzimologia , Humanos , Cinética , Biossíntese de ProteínasRESUMO
Transcription factor TFIIIC2 derived from human cells is required for tRNA-type gene transcription and binds with high affinity to the essential B-box promoter element of tRNA-type genes. Although 5S rRNA genes contain no homology with the tRNA-type gene B box, we show that TFIIIC2 is also required for Xenopus laevis 5S rRNA gene transcription. TFIIIC2 protected an approximately 30-base-pair (-10 to +18) region of a Xenopus 5S rRNA gene from DNase I digestion. This region, which spanned the transcription start site, included sequences that are highly conserved among eucaryotic 5S rRNA genes and have no homology with the B-box sequence of tRNA genes. Mutation of the TFIIIC2-binding site reduced transcription of the 5S rRNA gene by a factor of 10 in HeLa cell extracts. Methylation of C residues within the TFIIIC2-binding site interfered with binding of TFIIIC2. These results suggest a role of the TFIIIC2-binding sequence in 5S rRNA gene transcription. In addition, the 5S rRNA gene binding site and the tRNA-type gene B-box sequence did not compete with each other for binding to TFIIIC2 any better than did an unrelated DNA sequence, indicating that TFIIIC2 interacts with 5S rRNA genes and tRNA-type genes through separate DNA-binding domains or polypeptides.
Assuntos
DNA/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Citosina/metabolismo , DNA/genética , Desoxirribonuclease I/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Xenopus laevisRESUMO
Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Fatores de Transcrição/ultraestrutura , Sítios de Ligação , Proteínas Fúngicas/ultraestrutura , Expressão Gênica , Genes Fúngicos , Técnicas In Vitro , Estrutura Molecular , Mapeamento de Peptídeos , Regiões Promotoras Genéticas , Conformação Proteica , Proteínas Recombinantes , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Saccharomyces cerevisiae , Relação Estrutura-Atividade , Temperatura , Fator de Transcrição TFIID , Transcrição GênicaRESUMO
A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (approximately 500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (approximately 2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at approximately 500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 x 10(5) to 6 x 10(5) molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the approximately 2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The approximately 2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.
Assuntos
Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Extratos Celulares , Fracionamento Celular , Cromatografia em Gel , Ciclina C , Quinase 8 Dependente de Ciclina , Células HeLa , Humanos , Transcrição GênicaRESUMO
Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/química , Poliovirus/enzimologia , Fatores de Transcrição TFIII , Fatores de Transcrição/química , Proteínas Virais , Proteases Virais 3C , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/metabolismo , Poliomielite/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription. Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions. A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G. Kassavetis, C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek, Mol. Cell. Biol. 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment. Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription. These results indicate that Brf and TFIIB interact differently with TBP. An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs. Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.