RESUMO
We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCl or 1 mg of heparin per ml soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.
Assuntos
Actinas/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Drosophila , Heparina/farmacologia , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Cloreto de Potássio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica , Moldes Genéticos , Transcrição Gênica/efeitos dos fármacosRESUMO
We have purified factor 5, a Drosophila RNA polymerase II transcription factor. Factor 5 was found to be required for accurate initiation of transcription from specific promoters and also had a dramatic effect on the elongation properties of RNA polymerase II. Kinetic studies suggested that factor 5 stimulates the elongation rate of RNA polymerase II on a dC-tailed, double-stranded template by reducing the time spent at the numerous pause sites encountered by the polymerase. The factor was found to be composed of two polypeptides (34 and 86 kilodaltons). Both subunits bound tightly to pure RNA polymerase II but were not bound to polymerase in elongation complexes. Our results suggest that factor 5 interacts briefly with the paused polymerase molecules and catalyzes a conformational change in them such that they adopt an elongation-competent conformation.
Assuntos
Drosophila/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Animais , Drosophila/genética , Cinética , Conformação Proteica , Fatores de Transcrição/isolamento & purificação , Transcrição GênicaRESUMO
We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.
Assuntos
Actinas/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Drosophila , Heparina/farmacologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Cloreto de Potássio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA/biossíntese , RNA/isolamento & purificação , Mapeamento por Restrição , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Moldes GenéticosRESUMO
Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Biotina/metabolismo , Quinase 9 Dependente de Ciclina , Quinases Ciclina-Dependentes/efeitos dos fármacos , Diclororribofuranosilbenzimidazol/farmacologia , Inibidores Enzimáticos/farmacologia , Repetição Terminal Longa de HIV , Humanos , Fosforilação/efeitos dos fármacos , Fator B de Elongação Transcricional Positiva , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Especificidade por Substrato , Moldes Genéticos , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.
Assuntos
Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Peptidilprolil Isomerase/antagonistas & inibidores , DNA Recombinante , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Plasmídeos/genética , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Compostos de Sulfidrila/química , Transcrição Gênica/efeitos dos fármacosRESUMO
Cyclin T1 has been identified recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is sufficient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-specific signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the first time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.
Assuntos
Ciclinas/metabolismo , Ativação Linfocitária , Proteínas Quinases/metabolismo , Linfócitos T/metabolismo , Regulação para Cima , Ciclo Celular , Células Cultivadas , Ciclina T , Quinase 9 Dependente de Ciclina , HIV-1 , Células HeLa , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Mitógenos/farmacologia , Fosforilação , Fito-Hemaglutininas/farmacologia , RNA Polimerase II/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A phosphocellulose flowthrough fraction required for accurate transcription in vitro by RNA polymerase II was found to contain a DNase inhibitor which was necessary to maintain template integrity (Price D.H., Sluder A.E. & Greenleaf A.L. (1987) J. Biol. Chem. 262, 3244-3255). Starting with a Drosophila Kc cell nuclear extract, the DNase inhibitory activity has been purified 19,000-fold. In combination with the other necessary fractions, the highly purified inhibitor continues to support reconstruction of transcription. It thus appears to be the only required activity in the original phosphocellulose flowthrough fraction. The inhibitor is a protein which does not bind to DNA or inhibit DNase I, so that it has also been useful in assays for DNA binding proteins in crude, DNase-contaminated fractions.
Assuntos
Desoxirribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/metabolismo , Drosophila , Peso Molecular , Moldes GenéticosRESUMO
The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. TFAP2C is a transcription factor that has a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines. Comparing the genomic binding sites for TFAP2C, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knockdown of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5'-aza-2-deoxycytidine (5'-aza-dC) (an inhibitor of DNA methylation) allowed TFAP2C to bind to the GPX1 promoter resulting in the activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in â¼20% of primary breast cancers and a highly significant correlation between the TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells.
Assuntos
Neoplasias da Mama/genética , Ilhas de CpG/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/genética , Fator de Transcrição AP-2/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Decitabina , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Glutationa Peroxidase/metabolismo , Humanos , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fator de Transcrição AP-2/metabolismo , Transcrição Gênica , terc-Butil Hidroperóxido/farmacologia , Glutationa Peroxidase GPX1Assuntos
Cromossomos/fisiologia , Oócitos/fisiologia , Óvulo/fisiologia , Poli A/genética , RNA/genética , Animais , DNA Mitocondrial/genética , Feminino , Microscopia Eletrônica , Hibridização de Ácido Nucleico , Poli A/isolamento & purificação , RNA/isolamento & purificação , RNA Mensageiro , Sequências Repetitivas de Ácido Nucleico , XenopusRESUMO
Transcription elongation factor S-II mediates nascent transcript cleavage by RNA polymerase II (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have examined the mechanism of action of the Drosophila S-II analog, DmS-II, in a defined transcription system. Our results show that DmS-II is necessary and sufficient to activate nascent transcript cleavage by RNA polymerase II during transcription of a dC-tailed template. The pattern of transcripts resulting from prolonged action by DmS-II indicates that there are kinetic barriers to transcript shortening. During the cleavage reaction, the polymerase remains in register with the template strand and generates mainly nucleotide dimers. The ability of DmS-II to mediate transcript shortening resides in the carboxyl-terminal half of the protein. Our results support a model for pause suppression in which DmS-II binds to the paused polymerase, causes one cleavage event and is then released from the complex. Elongation by the polymerase then allows a second encounter with the pause site and a second chance of passing the site. Complete pause suppression may require multiple transcript shortening events for some polymerase molecules.
Assuntos
Drosophila/enzimologia , RNA Polimerase II/metabolismo , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Sítios de Ligação , Proteínas de Drosophila , Cinética , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , RNA/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ribonuclease H/metabolismo , Moldes Genéticos , Fatores de Transcrição/metabolismoRESUMO
Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC(50) determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Piperidinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Polimerase II/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Drosophila , Flavonoides/metabolismo , Piperidinas/metabolismo , Fator B de Elongação Transcricional Positiva , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Drosophila factor 2, an RNA polymerase II transcript release factor, exhibits a DNA-dependent ATPase activity (Xie, Z., and Price D. H. (1997) J. Biol. Chem. 272, 31902-31907). We examined the nucleic acid requirement and found that only double-stranded DNA (dsDNA) effectively activated the ATPase. Single-stranded DNA (ssDNA) not only failed to activate the ATPase, but suppressed the dsDNA-dependent ATPase. Gel mobility shift assays showed that factor 2 formed stable complexes with dsDNA or ssDNA in the absence of ATP. However, in the presence of ATP, the interaction of factor 2 with dsDNA was destabilized, while the ssDNA-factor 2 complexes were not affected. The interaction of factor 2 with dsDNA was sensitive to increasing salt concentrations and was competed by ssDNA. In both cases, loss of binding of factor 2 to dsDNA was mirrored by a decrease in ATPase and transcript release activity, suggesting that the interaction of factor 2 with dsDNA is important in coupling the ATPase with the transcript release activity. Although the properties of factor 2 suggested that it might have helicase activity, we were unable to detect any DNA unwinding activity associated with factor 2.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , DNA Helicases/metabolismo , Ligação Proteica , RNA Mensageiro/genéticaRESUMO
Factor 2 was previously identified in Drosophila Kc cell nuclear extract (KcN) as an activity suppressing the appearance of long transcripts (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). A 154-kDa protein with factor 2 activity was purified to apparent homogeneity from KcN. An immobilized template assay indicated that factor 2 caused the release of transcripts by RNA polymerase II in an ATP-dependent manner. Some early elongation complexes were resistant to factor 2 action but became sensitive after treatment with 1 M KCl. In the absence of factor 2, transcription complexes still exhibited a low degree of processivity suggesting that factor 2 was only partially responsible for abortive elongation.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Ribonucleotídeos/metabolismo , Supressão GenéticaRESUMO
Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H. (1992) Mol. Cell. Biol. 12, 2078-2090). We have fractionated nuclear extracts of Drosophila melanogaster Kc cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long run-off transcripts.
Assuntos
Diclororribofuranosilbenzimidazol/farmacologia , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica/efeitos dos fármacos , Animais , Sistema Livre de Células , Células Cultivadas , Drosophila melanogaster/citologia , Regulação da Expressão Gênica , Conformação Proteica , Fatores de Transcrição/metabolismoRESUMO
In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.
Assuntos
Acanthamoeba/metabolismo , Nucleotídeos/metabolismo , Edição de RNA/genética , RNA de Transferência/metabolismo , RNA/genética , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Endonucleases/metabolismo , Escherichia coli/genética , Exonucleases/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mitocondrial , RNA de Transferência de Leucina/genética , RNA de Transferência de Tirosina/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Transcrição Gênica/genéticaRESUMO
In the amoeboid protozoon Acanthamoeba castellanii 13 of the 16 mtDNA-encoded tRNA sequences have mis-matches at one or more of the first three positions in the acceptor stem. A previous study had indicated that these mis-matches are corrected by a form of RNA editing. In the present study, the pattern of editing was further investigated by sequence analysis of both halves of the acceptor stem of 11 mtDNA-encoded tRNAs. The results confirm all of the remaining editing sites predicted on the basis of the secondary structure modelling of A. castellanii mitochondrial tRNAs, and identify two unexpected edits. We also investigated the expression and editing of transcripts of an unusual trnX gene specifying an eight-nucleotide anticodon loop sequence. Although no mature 3'-CCAOH-containing tRNAX products were detected, editing was observed in some circularized tRNAX clones. The implications of these results with respect to the mechanism of editing and the evolutionary origin of this process are discussed.
Assuntos
Acanthamoeba/genética , Edição de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , RNA/genética , Animais , Sequência de Bases , Primers do DNA/genética , DNA Mitocondrial/genética , DNA de Protozoário/genética , Evolução Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mitocondrial , RNA de Protozoário/genética , RNA de Transferência/química , Análise de Sequência de RNARESUMO
We have examined the process by which the 3' terminus of the Drosophila histone H3 mRNA is produced in vitro. When a template containing a portion of DNA that flanks the normal 3' end of the histone H3 gene and an oligo dC tail on the template strand is transcribed in vitro by Drosophila RNA polymerase II, transcription continues beyond the 3' end of the H3 gene. A processing activity was identified that cleaves the precursor transcript generating an RNA species with the same 3' end as the mature H3 mRNA. The processing activity was partially purified by ion exchange chromatography and sucrose gradient sedimentation. The isolated activity was found to require Mg++ but did not require addition of a nucleoside triphosphate for activity. The activity sedimented with a molecular weight of approximately 140,000 daltons. Transcription of the template and processing of the RNA can be uncoupled in vitro.
Assuntos
Drosophila melanogaster/genética , Histonas/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Animais , Sistema Livre de Células , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
RNA polymerase II will efficiently initiate transcription on linear duplex DNA which has been extended at its 3' ends by the addition of short stretches of polydeoxycytidine (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295). We have used such dC-tailed templates to identify factors affecting elongation by Drosophila RNA polymerase II (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). While studying these factors we have observed two unexpected characteristics of transcription of the tailed templates. First, we found that RNA polymerase II encountered a strong pause site after the incorporation of 14 nucleotides. This pausing was observed on all templates examined and with RNA polymerase II from a variety of sources. In addition, we found that ammonium ions markedly stimulated the polymerase, increasing both the efficiency with which the enzyme left the 14 base pause site and the subsequent rate of elongation. A factor previously shown to affect transcription of dC-tailed templates (factor 4, Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255) was found to cause transcript displacement and to stimulate the elongation rate approximately 2-fold. This factor copurified with an RNase H activity, and a model is presented for the mechanism of transcript displacement by RNase H. The observations presented here form a basis for further analysis of RNA polymerase II elongation and its modulation by transcription factors. They should also aid in the interpretation of other experiments in which dC-tailed templates are used.
Assuntos
DNA/metabolismo , Drosophila/enzimologia , RNA Polimerase II/metabolismo , Transcrição Gênica , Sulfato de Amônio/farmacologia , Animais , Desoxicitidina/metabolismo , Endorribonucleases/metabolismo , Cinética , Ácidos Nucleicos Heteroduplexes/metabolismo , Nucleotídeos/metabolismo , Cloreto de Potássio/farmacologia , RNA/genética , RNA/metabolismo , Ribonuclease H , Ribonuclease Pancreático/metabolismo , Moldes Genéticos , Transcrição Gênica/efeitos dos fármacosRESUMO
Drosophila Kc cells were utilized to prepare nuclear extracts in which promoter-containing DNA templates were efficiently transcribed by RNA polymerase II. A combination of fractionation schemes was used to identify and partially purify seven activities (factors) which affected the transcription of four different genes in vitro. Reconstructing specific transcription required exogenous RNA polymerase II in addition to these factors. Moreover, the high efficiency of transcription characteristic of the crude extract was preserved in reconstruction reactions. The methods used are presented in detail. Functions were assigned to several of the factors. One essential factor appeared to affect initiation and displayed chromatographic properties unlike any other Drosophila transcription factor previously described. Two factors specifically affected RNA chain elongation. Another activity was a DNase inhibitor required to preserve template integrity in the fractionated system. The remaining three factors were not absolutely essential but affected the specific in vitro transcription either qualitatively or quantitatively. A comparison of these transcription factors with other Drosophila and mammalian transcription factors is made.