In-vitro competition analysis of procyclin gene and variant surface glycoprotein gene expression site transcription in Trypanosoma brucei

In Trypanosoma brucci, alpha -amanitin-resistant transcription characterist ic of RNA polymerase I is initiated at ribosomal RNA gene (RRNA), procyclin gene (GPEET or EP1), and variant surface glycoprotein gene expression site (VSG ES) promoters. The three promoter types do not share obvious sequence homologies, but contain a proximal domain I and a distal domain II within 80 bp upstream of the transcription initiation site. RRNA, GPEET and EP1, b ut not the VSG ES promoter, require additional upstream sequences for full activity. In the present study, we competed in-vitro transcription of circu lar template DNA with linear DNA fragments to identify promoter domains res ponsible for binding and sequestering essential trans-acting transcription factors. For the GPEET promoter, we found that domain III, located between positions - 141 and - 92, was most important for the DNA fragment to exert a transcription competition effect, whereas domain I, the only element abso lutely required for transcription, was not. Moreover, insertions between pr omoter domains II and III reduced both transcription from the GPEET promote r and competition with the GPEET promoter fragment, suggesting that these t wo domains cooperate in the formation of a stable DNA-protein complex. Take n together, these results indicate a promoter structure very similar to tha t of the Saccharomyces cerevisiae RRNA promoter. In contrast, VSG ES promot er analysis showed that domains I and II are both necessary and sufficient to compete transcription. Despite this structural difference, our analysis provide evidence that GPEET and VSG ES promoters interact with a common fac tor that is also important for RRNA promoter transcription. (C) 2001 Elsevi er Science B.V. All rights reserved.