Characterization of the RNA polymerases of Crithidia fasciculata

We have characterized the crithidial RNA polymerases (RNAPs). The RNAPs from this organism were resolved by chromatography and could be classified into RNAP I, II and III because of their differential sensitivity to class-specific RNA polymerase inhibitors, such as alpha-amanitin (ama) and tagetitoxin. The three RNAP classes were subsequently characterized in nuclear run-ons using class-specific DNA templates and the inhibitory effect of ama on RNA chain elongation. These experiments showed that Crithidia fasciculata contains the normal set of eukaryotic RNAPs. However, RNAP II was found to be relatively resistant to ama, which seems to be a general feature of kinetoplastid parasites. Tagetitoxin, a potent inhibitor of purified crithidial RNAP III, does not inhibit RNAP III in nuclear run-on experiments, suggesting that the inhibitory effect of tagetitoxin depends on the enzyme conformation, i.e. 'naked' enzyme versus that in a transcription complex. Finally, the role of RNAP III in the transcription of the mini-exon genes, which provide the 5' end of each mRNA, is discussed.