TESTING PROMOTER ACTIVITY IN THE TRYPANOSOME GENOME - ISOLATION OF A METACYCLIC-TYPE VSG PROMOTER, AND UNEXPECTED INSIGHTS INTO RNA-POLYMERASE-II TRANSCRIPTION

In trypanosomes, most genes are arranged in polycistronic transcriptio n units. Individual mRNAs are generated by 5'-trans splicing and 3' po lyadenylation. Remarkably, no regulation of RNA polymerase II transcri ption has been detected although many RNAs are differentially expresse d during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vec tors that were designed to allow the detection of low promoter activit ies in a transcriptionally silent chromosomal context, we isolated a n ovel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcr iption was a-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favor ed by genomic accessibility and double-strand melting. (C) 1998 Academ ic Press.