In Vitro Analysis of α-Amanitin-Resistant Transcription from the rRNA, Procyclic Acidic Repetitive Protein, and Variant Surface Glycoprotein Gene Promoters in Trypanosoma brucei

Abstract

In Trypanosoma brucei, transcription resistant to the mushroom toxin α-amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and α-amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn²⁺. Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency.

ACKNOWLEDGMENTS

G. Laufer and G. Schaaf contributed equally to this work.

We are grateful to Christine Clayton and Etienne Pays for sending us their gene constructs, to Michael Duszenko for providing us with the procyclic T. brucei cell line, and to Albrecht Bindereif and Laurence Barker for critically reading the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft (grant Gu 371/3-1).