In vitro analysis of alpha-amanitin-resistant transcription from the rRNA,procyclic acidic repetitive protein, and variant surface glycoprotein genepromoters in Trypanosoma brucei

In Trypanosoma brucei transcription resistant to the mushroom toxin ar-aman itin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, b ut extends to genes encoding the major cell surface proteins variant surfac e glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein ( PARP). Here, we report the development of a homologous cell extract from pr ocyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters dri ve efficient, accurate, and or-amanitin-resistant transcription. A comparat ive analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheles s, PARP promoter transcription proved to be exceptional by its high efficie ncy, its lag phase, a high template DNA concentration optimum, and its tole rance to increasing concentrations of Mn2+. Mutational analysis for both th e PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstr eam control regions (UCRs), however, had a different effect. Whereas PARP U CR deletion reduced transcription efficiency almost 10-fold, deletion of th e rDNA UCR had only a minor effect on transcription efficiency.