RESUMO
Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.
Assuntos
Candida albicans/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Compartimento Celular , Núcleo Celular/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Teste de Complementação Genética , Biblioteca Genômica , Dados de Sequência Molecular , Mutação , Proteínas Nucleares , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Telômero/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas rap de Ligação ao GTPRESUMO
A Candida albicans gene encoding a novel DNA-binding protein that bound to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans was previously cloned and designated RBF1 (RPG-box-binding factor). In this report, determination of the functional domains of the protein is described. The DNA-binding domain was 140 aa in length, was centrally located between two glutamine-rich regions, and correlated with transcriptional activation in S. cerevisiae. The results, together with the previous finding that showed its predominant localization in the nucleus, suggest that this DNA-binding protein could be a transcription factor. Disruption of the functional RBF1 gene of C. albicans strains caused an alteration in cell morphology to the filamentous form on all solid and liquid media tested. Thus, we speculate that Rbf1p may be involved in the regulation of the transition between yeast and filamentous forms at the level of transcription.
