Characterization of a TBP-like factor from Trypanosoma cruzi

CRIBB, PAMELA; ZELUS, DOMINIQUE; ZEMZOUMI, KHALID; SERRA, ESTEBAN

Caxambu. MG. Brasil

Congreso; XXIX Annual Meeting on Basic Research in Chagas Disease- XVIII Meeting of the Brazilian Society of Protozoology; 2002

Brazilian Society of Protozoology.

CHARACTERIZATION OF A TBP-LIKE FACTOR FROM Trypanosoma cruzi. Cribb, P.; Zelus, D.; Zemzoumi, K. & Serra, E.      Instituto de Biología Molecular y Celular de Rosario. Rosario. Argentina.      Pcribb@fbioyf.unr.edu.      TATA-binding protein (TBP) is an ubiquitous transeription factor that takes part of transeription Initiation Complex (IC) of all type-I, -II and –III eukariotic RNA polymerases. In the last years, a regulatory function of IC, vía regulatory TAFs and alternative TBP-Iike factars (TLFs), was established in yeasts and metazoans. Using degenerated oligonueleotides a TLF from Trypanosoma cruzi was cloned by PCR(TcTLF-1). TcTLF-1 is coded by an unique gene in all T. cruzi strains assayed, being highly conserved at the sequence level among all them. Deduced TcTLF-1 sequence showed low, but significant identity with all TBPs, TLFs and archaeal TBPs. Sequence analysis determined that Tc-TLF-1 presents intermediate characteristics between those from canonical TBPs and alternative TLFS. The coding sequence for TcTLF-1 was cioned in expression vectors (pGEX and pQE)providing recombinant proteins both as a fussion to GST and with Histidine tail. Policional antibodies against recombinant protein were used in Western Blot assays indicating that the protein is present in both epimastigotes and trypomastigotes. Electrophoretic mobility shift assays showed that recombinant TcTLF-1 binds to the SL RNA gene promoter, the unique RNA pol II-dependent promoter identified in trypanosomatids. Moreover, the presence of TcTLF- 1 in nuclear proteins- DNA complexes was demonstrated by super shift assays.Finally, "Selex" assays suggest that recombinant TcTLF- 1 binds preferably to a consensus sequence rich in G+C, similar to those present in the SL RNA gene promoter. Since no RNApol II-dependent transcriptional regulation mechanism has been described in trypanosomatids to date, we propose that gene expression may be regulated at the transcriptional level by TcTLF-1 through the IC.