A TFIIH-Associated Mediator Head Is a Basal Factor of Small Nuclear Spliced Leader RNA Gene Transcription in Early-Diverged Trypanosomes

Abstract

Genome annotation suggested that early-diverged kinetoplastids possess a reduced set of basal transcription factors. More recent work, however, on the lethal parasite Trypanosoma brucei identified extremely divergent orthologs of TBP, TFIIA, TFIIB, and TFIIH which, together with the small nuclear RNA-activating protein complex, form a transcription preinitiation complex (PIC) at the spliced leader (SL) RNA gene (SLRNA) promoter. The SL RNA is a small nuclear RNA and a trans splicing substrate for the maturation of all pre-mRNAs which is metabolized continuously to sustain gene expression. Here, we identified and biochemically characterized a novel TFIIH-associated protein complex in T. brucei (Med-T) consisting of nine subunits whose amino acid sequences are conserved only among kinetoplastid organisms. Functional analyses in vivo and in vitro demonstrated that the complex is essential for cell viability, SLRNA transcription, and PIC integrity. Molecular structure analysis of purified Med-T and Med-T/TFIIH complexes by electron microscopy revealed that Med-T corresponds to the mediator head module of higher eukaryotes. These data therefore show that mediator is a basal factor for small nuclear SL RNA gene transcription in trypanosomes and that the basal transcription function of mediator head is a characteristic feature of eukaryotes which developed early in their evolution.

Supplemental material for this article may be found at http://mcb.asm.org/.

This study was funded by NIH grants AI073300 to A.G., GM67167 to F.J.A., and AI26756 and AI29735 to J.D.R. J.H.L. was supported by KOSEF grant C00093. S.D.E. is the recipient of a career development award from the Northeastern Research Center for Excellence (NIH grant U54 AI057159).

We thank Nicolai Siegel for advice on ChIP assays.