RESUMO
A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5'-mRNA cap, i.e., m(7)GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m(7)GMP (or m(2,7)GMP) as one of the reaction products. Interestingly, m(7)Gpppm(3)(N6, N6, 2'O)A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m(7)Gpppm(3)(N6, N6, 2'O)Apm(2'O)Apm(2'O)Cpm(2)(N3, 2'O)U, that occurs in these organisms.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Hidrolases Anidrido Ácido/genética , Sequência de Aminoácidos , Animais , Fosfatos de Dinucleosídeos/metabolismo , Genes de Protozoários , Cinética , Metilação , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Protozoários/genética , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma brucei brucei/genéticaRESUMO
Protein-coding genes of trypanosomes are mainly transcribed polycistronically and cleaved into functional mRNAs in a process that requires trans splicing of a capped 39-nucleotide RNA derived from a short transcript, the spliced-leader (SL) RNA. SL RNA genes are individually transcribed from the only identified trypanosome RNA polymerase II promoter. We have purified and characterized a sequence-specific SL RNA promoter-binding complex, tSNAP(c), from the pathogenic parasite Trypanosoma brucei, which induces robust transcriptional activity within the SL RNA gene. Two tSNAP(c) subunits resemble essential components of the metazoan transcription factor SNAP(c), which directs small nuclear RNA transcription. A third subunit is unrelated to any eukaryotic protein and identifies tSNAP(c) as a unique trypanosomal transcription factor. Intriguingly, the unusual trypanosome TATA-binding protein (TBP) tightly associates with tSNAPc and is essential for SL RNA gene transcription. These findings provide the first view of the architecture of a transcriptional complex that assembles at an RNA polymerase II-dependent gene promoter in a highly divergent eukaryote.