Trypanosomal TBP Functions with the Multisubunit Transcription Factor tSNAP To Direct Spliced-Leader RNA Gene Expression

Abstract

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.

ACKNOWLEDGMENTS

We thank all Bellofatto lab members for comments on the manuscript; Chris Utter for help with antibody production; Joseph Milone for the control cell line; Martin Marinus for helpful discussions; Hong Li for mass spectrometry analysis; Douglass Lamont, David Martin, and Mike Ferguson for making available the TrypPEP database; Sara Melville for providing chromosome X shotgun sequencing clones; and the Tri-Trypanosome Genome Projects for their accessible data.

This work was supported by NIH grants AI29478 to V.B. and AI21729 to G.A.M.C. and American Heart Association Postdoctoral Fellowship 0425791T to J.B.P.