RESUMO
We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNA(Tyr) internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAP1 (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap1-1 mutant than in the wild type. The tap1-1 mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap1-1 mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts- growth defect, we cloned the wild-type TAP1 gene. It is essential for yeast growth. We show in the accompanying report (T. L. Aldrich, G. Di Segni, B. L. McConaughy, N. J. Keen, S. Whelen, and B. D. Hall, Mol. Cell. Biol. 13:3434-3444, 1993) that TAP1 is identical to RAT1, a yeast gene implicated in poly(A)+ RNA export and that the TAP1/RAT1 gene product has extensive sequence similarity to the protein encoded by another yeast gene (variously named DST2, KEM1, RAR5, SEP1, or XRN1) having exonuclease and DNA strand transfer activity (reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]).
Assuntos
Exorribonucleases , Proteínas Fúngicas/genética , Genes Fúngicos , Regiões Promotoras Genéticas , RNA de Transferência de Tirosina/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Alelos , Ligação Competitiva , Clonagem Molecular , Cruzamentos Genéticos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas Fúngicas/biossíntese , Mutação , Plasmídeos , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Fúngico/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/crescimento & desenvolvimento , Supressão Genética , Transcrição GênicaRESUMO
Sequence data are presented for the Saccharomyces cerevisiae TAP1 gene and for a mutant allele, tap1-1, that activates transcription of the promoter-defective yeast SUP4 tRNA(Tyr) allele SUP4A53T61. The degree of in vivo activation of this allele by tap1-1 is strongly affected by the nature of the flanking DNA sequences at 5'-flanking DNA sequences as far away as 413 bp from the tRNA gene and by 3'-flanking sequences as well. We considered the possibility that this dependency is related to the nature of the chromatin assembled on these different flanking sequences. TAP1 encodes a protein 1,006 amino acids long. The tap1-1 mutation consists of a thymine-to-cytosine DNA change that changes amino acid 683 from tyrosine to histidine. Recently, Amberg et al. reported the cloning and sequencing of RAT1, a yeast gene identical to TAP1, by complementation of a mutant defect in poly(A) RNA export from the nucleus to the cytoplasm (D. C. Amberg, A. L. Goldstein, and C. N. Cole, Genes Dev. 6:1173-1189, 1992). The RAT1/TAP1 gene product has extensive sequence similarity to a yeast DNA strand transfer protein that is also a riboexonuclease (variously known as KEM1, XRN1, SEP1, DST2, or RAR5; reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). The tap1-1 amino acid substitution affects a region of the protein in which KEM1 and TAP1 are highly similar in sequence.
Assuntos
DNA Fúngico/metabolismo , Exorribonucleases , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , RNA de Transferência de Tirosina/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
The optimum monovalent cation concentration (Na+ or K+) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150-200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalent cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking -closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/metabolismo , DNA Viral/metabolismo , Potássio/farmacologia , Sódio/farmacologia , Animais , Cátions Monovalentes , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Fígado/enzimologia , Camundongos , Ratos , Vírus 40 dos SímiosAssuntos
DNA , Nucleotídeos , Animais , Isótopos de Carbono , Celulose , Fenômenos Químicos , Físico-Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Colífagos , Cricetinae , DNA/metabolismo , DNA Bacteriano , DNA Viral , Dextranos , Escherichia coli , Células L , Camundongos , Desnaturação de Ácido Nucleico , Isótopos de Fósforo , Temperatura , Cultura de VírusRESUMO
When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
Assuntos
DNA Bacteriano/biossíntese , DNA Viral/biossíntese , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Vírus 40 dos Símios/metabolismo , Centrifugação com Gradiente de Concentração , Estabilidade de Medicamentos , Temperatura Alta , Peso Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Moldes GenéticosRESUMO
The DNA untwisting enzyme has been purified approximately 300-fold from rat liver nuclei. The protein is greater than 90% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis. The native enzyme has a molecular weight between 64 000 and 68 000 and is composed of a single polypeptide chain. Evidence is presented that the protein can act catalytically. A trace amount of endonuclease activity associated with the most pure fraction could be a contaminant or it could be due to the action of the DNA untwisting enzyme itself.
Assuntos
Desoxirribonucleases/isolamento & purificação , Fígado/enzimologia , Polinucleotídeo Ligases/isolamento & purificação , Animais , DNA Circular , DNA Viral , Desoxirribonucleases/metabolismo , Cinética , Polinucleotídeo Ligases/metabolismo , Ratos , Vírus 40 dos Símios , Espectrometria de FluorescênciaRESUMO
Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1%. The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.
Assuntos
Clonagem Molecular , DNA/análise , Plasmídeos , Saccharomyces cerevisiae/genética , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , Escherichia coli/genética , Teste de Complementação Genética , Poli A/genética , RNA/genética , RNA MensageiroRESUMO
The C-terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle. The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms. Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all. The functional and evolutionary implications of this variation among RPB1 C-termini is largely unexplored. We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild-type CTD. The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad. Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress. These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD.
Assuntos
Evolução Biológica , RNA Polimerase II/química , Amoeba/enzimologia , Animais , Fosforilação , RNA Polimerase II/fisiologia , Sequências Repetitivas de Aminoácidos , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , TemperaturaRESUMO
Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.