Transcription regulation in Protists. Cloning of a TBP-like factor from Trypanosoma cruzi.

CRIBB, PAMELA; ZELUS, DOMINIQUE; TROCHINE, ANDREA; ZEMZOUMI, KHALID; LUJAN, HUGO; GIRARDINI, JAVIER; SERRA, ESTEBAN

Woods Hole, Ma. USA

Congreso; XIII Molecular Parasitology Meeting; 2002

Marine Biological Laboratory, Woods Hole, Ma. USA

Transcription regulation in Protists. Cloning of a TBP-like factor from Trypanosoma cruzi. Pamela Cribb, Dominique Zelus, Andrea Trochine, Khalid Zemzoumi, Hugo Lujan, Javier Girardini and Esteban Serra. TATA-binding protein (TBP) is an ubiquitous transcription factor that takes part of transcription Initiation Complex (IC) of all type-I, -II and -III RNA polymerases. In the last years, a regulatory function of IC, via regulatory TAFs and alternative TBP-like factors (TLFs), was established in yeasts and metazoans. Using degenerated oligonucleotides a TLF from Trypanosoma cruzi (CL Brener) was cloned by PCR (TcTLF). TcTLF is coded by an unique gene in all T. cruzi strains assayed, being highly conserved at the sequence level among all them. Deduced Tc-TLF sequence showed low, but significant, identity with all TBPs, TLFs and archaeal TBPs. Sequence analysis determined that Tc-TLF shares characteristic with both canonical TBPs and alternative TLFs. Recombinant TcTLF was obtained as GST and his-tag fusion protein, purified and used to obtain policlonal antibodies. Western blot experiments indicates that Tc-TLF is highly accumulated in epimastigotes in comparison to trypomastigotes. The presence of TcTLF in protein complexes interacting with RNA pol II-dependent SL RNA promoter was revealed by super shift analysis. Recombinant Tc-TLF was used in “Selex” assays in order to determine its binding sequence preferences. By this way, we obtained a consensus sequence rich in G+C. Multiple Expectation maximization for Motif Elicitation analysis determined the existence at least one motif related to that obtained by selex in SL RNA and ribosomal promoters from several T. cruzi strains. By analysis of Trypanosomes and Giardia genome databases we detected several IC putative components, including some of those described as regulatory in yeasts and metazoans. Our results leads us to hypothesize the existence in Protists of a simplified eukariotic-like IC able to mediate transcription regulation.