Basal Nuclear factors from Trypanosoma cruzi

CRIBB PAMELA; TROCHINE ANDREA; ZELUS DOMINIQUE; SERRA ESTEBAN

Iguazú, Argentina

Simposio; SYMPOSIUM "GENE EXPRESSION AND RNA PROCESSING"; 2003

THE INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY

Gene Expression and RNA Processing. INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY (ICGEB) MEETING. Iguazu. 2003. Basal Nuclear Factors from Trypanosoma cruzi. Pamela Cribb; Andrea Trochine; Dominique Zelus and Esteban Serra. Instituto de Biologia Molecular y Celular de Rosario. Facultad de Ciencias Bioquimicas y Farmacéuticas. Suipacha 531. CP 2000 Rosario. Argentina. Transcription regulation mechanisms remain completely unknown in Trypanosomatids. TBP is an ubiquitous transcription factor that takes part of transcription Initiation Complex (IC) of all type-I,   -II and -III RNApolymerases. TBPs and their Archaeal counterparts, are the cornerstone for building up protein complexes that mediate the landing of polymerases on promoters. We propose that assembly of these protein complexes, and its interaction with the RNA polymerases, chromatin proteins and DNA, could be the major regulatory events at the transcription initiation level in Trypanosomatids. Using degenerated oligonucleotides a TBP-Like Factor from Trypanosoma cruzi (TcTLF) was cloned. TcTLF is coded by a unique gene in all strains assayed, being highly conserved among all them. A phylogenetic analysis grouped TcTLF together with proteins from other deep-branching protists in a clade that coincides with a CTD-less RNA polymerase II clade recently described. This clade contains organisms lacking trans-regulatory factors, that should have regulatory mechanisms different from those found in Fungi, Metazoans and Plants. TcTLF is present in both epimastigotes and trypomastigotes as determined by western blot. “Selex” assays using recombinant TcTLF determined an atipical GC-rich consensus binding site for this protein. The presence of TcTLF in protein complexes interacting with RNA pol II-dependent Spliced Leader (SL) RNA promoter was revealed by super shift analysis. Recombinant protein also interact with this probe. Genome project available data represent more than 10 times the T. cruzi genome content. 973,418 single pass sequences were analized using Blast and profile-sensitive search programs. A unique TFIIB-related sequence was detected in this data. When analyzed, this sequence grouped, together with those from the CDT-less clade organisms, in a subclade of BRFs. No canonical TFIIB sequence was identified in this organisms. Finally, several sequences sharing significative similarity with Histone Fold Motif and Bromodomains, typical of IC componets, were detected in T. cruzi genome.