RESUMEN
We present a physical and molecular genetic characterization of Drosophila melanogaster TFIIE (dTFIIE), a component of the basal RNA polymerase II transcription apparatus. We have purified dTFIIE to near homogeneity from nuclear extracts of Drosophila embryos and found that it is composed of two subunits with apparent molecular weights of 55 and 38 kDa. Peptide sequence information derived from the two subunits was used to isolate the corresponding cDNA clones, revealing that dTFIIE and human TFIIE share extensive amino acid similarity. Functional conservation was demonstrated by the ability of bacterially expressed dTFIIE to substitute for human TFIIE in an in vitro transcription assay reconstituted from purified components. Cytological mapping analysis shows that both subunits are encoded by single copy genes located on chromosome III.
Asunto(s)
Drosophila melanogaster/química , Factores de Transcripción TFII , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Proteínas Recombinantes , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We show that the mammalian transcription Sp1 stimulates accurate transcription in a partially fractionated RNA polymerase II-dependent system from Drosophila cultured cells. Moreover, the extent of stimulation is equal for intact RNA polymerase II (polymerase IIA) and polymerase lacking the unique carboxyl-terminal domain of the largest subunit (polymerase IIB). We conclude that in this system Sp1 interacts with a component of the transcription machinery, other than the carboxyl-terminal domain, which is preserved between mammals and insects.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/genética , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Drosophila/enzimología , Drosophila/genética , Proteínas de Choque Térmico/genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factor de Transcripción Sp1 , Transcripción GenéticaRESUMEN
We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the beta' subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the alpha-amanitin-resistant allele, RpII215C4, includes all sequences required to produce amanitin-resistant transformants.
Asunto(s)
Drosophila melanogaster/genética , Genes , ARN Polimerasa II/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Drosophila melanogaster/enzimología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
DNA sequence analysis of RpII215, the gene that encodes the Mr215,000 subunit of RNA polymerase II (EC 2.7.7.6) in Drosophila melanogaster, reveals that the 3'-terminal exon includes a region encoding a C-terminal domain composed of 42 repeats of a seven-residue amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A hemi- and homozygous lethal P-element insertion into the coding sequence of this domain causes premature translation termination and therefore truncation of the protein, leaving only 20 heptamer repeats. While loss of approximately 50% of the repeat structure in this mutant is a lethal event in vivo, enzyme containing the truncated subunit remains capable of accurate initiation at promoters in vitro. Moreover, treatment of purified intact RNA polymerase II with protease, to remove the entire repeat domain, does not eliminate the enzyme's ability to initiate accurately in vitro. Possible in vivo functions for this unusual protein domain are considered in light of these results.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Relación Estructura-ActividadRESUMEN
P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.
Asunto(s)
Ritmo Circadiano , ADN/genética , Mutación , Transformación Genética , Cromosoma X , Animales , Conducta Animal , Mapeo Cromosómico , Drosophila melanogaster/genética , Femenino , Genotipo , Locomoción , Masculino , Linaje , PlásmidosRESUMEN
Mutations at the period (per) locus of Drosophila melanogaster disrupt several biological rhythms. Molecular cloning of DNA sequences encompassing the per+ locus has allowed germ-line transformation experiments to be carried out. Certain subsegments of the per region, transduced into the genome of arrhythmic pero flies, restore rhythmicity in circadian locomotor behavior and the male's courtship song.
Asunto(s)
Ritmo Circadiano , Drosophila melanogaster/genética , Mutación , Transformación Genética , Animales , Clonación Molecular , Enzimas de Restricción del ADN , Drosophila melanogaster/fisiología , Femenino , Genes , Masculino , Fenotipo , Conducta Sexual Animal , Vocalización AnimalRESUMEN
We have isolated and analyzed DNA sequences encompassing the period (per) locus of Drosophila melanogaster. The location of this clock gene was delimited by the molecular mapping of chromosome aberrations at or very near the per locus. At least five RNAs are transcribed from this region. One of these transcripts, a 0.9 kb species, is strongly implicated in per's control of biological rhythms. Two independently isolated arrhythmic mutations at the per locus dramatically reduce the level of this transcript. Furthermore, the level of the 0.9 kb transcript is strongly modulated during a light/dark cycle. We discuss evidence, from previously reported genetic and phenotypic analysis of per's function, suggesting that this region may be complex and that several gene products from the per region, including this 0.9 kb transcript, may be involved in the different aspects of normal rhythmicity influenced by this clock gene.
Asunto(s)
Drosophila melanogaster/genética , Mutación , Periodicidad , Transcripción Genética , Animales , Mapeo Cromosómico , Clonación Molecular , Drosophila melanogaster/fisiología , Larva/fisiología , Hibridación de Ácido Nucleico , Plásmidos , Pupa/fisiología , Translocación GenéticaRESUMEN
The soluble components of the RNA polymerase activity (N4 RNA polymerase II) required for coliphage N4 middle RNA synthesis have been purified to homogeneity using a complementation assay described elsewhere. These soluble components are found to exhibit the properties of a DNA-dependent RNA polymerase which is resistant to both rifampicin and streptolydigin and transcribes denatured N4 DNA with marked preference but with little selectivity for the middle region of the N4 genome. In its native form, the activity is composed of one Mr = 40,000 polypeptide (p4, the product of N4 cistron 4) and one Mr = 30,000 polypeptide (p7, the product of N4 cistron 3). Its physical properties and the mechanism of transcription selectivity are discussed.
Asunto(s)
Colifagos/enzimología , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Escherichia coli/enzimología , ARN Polimerasa II/aislamiento & purificación , Cinética , Sustancias Macromoleculares , Peso Molecular , Hibridación de Ácido Nucleico , ARN Polimerasa II/metabolismo , Moldes GenéticosRESUMEN
The requirement of a third gene product of molecular weight 15,000 for coliphage N4 middle RNA synthesis is described. Therefore, three proteins of molecular weights 15,000, 30,000, and 40,000, which constitute the N4 RNA polymerase II activity, are required for N4 middle RNA synthesis. A complementation assay for N4 RNA polymerase II activity, which requires a DNA-membrane complex carrying the 15,000-MW protein and a supernatant providing the 30,000- and 40,000-MW proteins, has been developed. This assay, which reproduces the requirement for the cistron 5 gene product in vitro provides a means for the purification of the soluble components of the N4 RNA polymerase II activity (to be reported elsewhere). The cistron 5 gene product is also required for N4 RNA polymerase II to synthesize RNAs utilizing middle promoters resident in recombinant plasmids. Mechanisms by which the cistron 5 gene product affects N4 RNA polymerase II transcription in vivo and in vitro are discussed.
Asunto(s)
Colifagos/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Genes Bacterianos , Genes Virales , Genes , ARN Polimerasa II/metabolismo , Transcripción Genética , Prueba de Complementación Genética , Cinética , Hibridación de Ácido Nucleico , PlásmidosAsunto(s)
Colifagos/genética , Expresión Génica , Biosíntesis de Proteínas , ARN Viral , Transcripción Genética , Enzimas de Restricción del ADN , ADN Viral , ARN Polimerasas Dirigidas por ADN/genética , Electroforesis en Gel de Agar , Escherichia coli/enzimología , Escherichia coli/genética , Genes Virales , Mutación , Ácidos Nucleicos Heterodúplex/biosíntesis , ARN Viral/biosíntesis , Rifampin/farmacología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
A DNA-dependent RNA polymerase has been purified to homogeneity from bacteriophage N4 virions. Sodium dodecyl sulfate-8 M urea polyacrylamide gel electrophoresis of the enzyme revealed a single polypeptide with a molecular weight of 350,000. The hydrodynamic properties of the enzyme have been determined to be 9.5 S for the sedimentation coefficient and 84 A for the Stokes radius. These two parameters indicate a native molecular weight of 320,000. Enzyme activity is dependent on the presence of Mg2+, the four ribonucleoside triphosphates, and denatured N4 DNA. Under these conditions, initiation of RNA synthesis occurs exclusively with pppG. The enzyme is inhibited by monovalent salts and is resistant to the drugs rifampicin and streptolydigin. The protein is present in 1 to 2 copies per virion; its presence provides an explanation for the independence of N4 early RNA synthesis on the activity of the host RNA polymerase.