RESUMO
Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA de Protozoário/genética , Telomerase/genética , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genética , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , DNA Recombinante/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dosagem de Genes , Genes de Protozoários/genética , Peso Molecular , Mutação/genética , RNA Polimerase III/metabolismo , RNA de Protozoário/análise , RNA Nuclear Pequeno/genética , Elementos de Resposta/genética , Telomerase/metabolismo , Moldes Genéticos , Tetrahymena thermophila/citologia , Tetrahymenina/enzimologia , Tetrahymenina/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.
