Physical and transcriptional analysis of the Trypanosoma brucei genome reveals a typical eukaryotic arrangement with close interspersionof RNA polymerase II- and III-transcribed genes.

To further our understanding of the structural and functional organization of the Trypanosoma brucei genome, we have searched for and analyzed sites in the genome where Pol II transcription units meet Pol III genes. Physical and transcriptional maps of cosmid clones spanning the Pol III-transcribed U2 small nuclear RNA (snRNA) and U3 snRNA/7SL RNA gene loci demonstrated that single-copy Pol II genes are closely associated with Pol III-transcribed genes, being separated from each other by 0.6-3 kb. At the U3/7SL transcriptional domain, two Pol II transcription units converged from either side of the chromosome towards the Pol III genes, suggesting that at least for the chromosome containing the U3 snRNA and 7SL RNA genes, there exist two distinct initiation sites for Pol II. Furthermore, in all cases the Pol III genes hallmark the end of Pol II transcription units, suggesting perhaps a functional role for this genetic arrangement. Lastly, we asked whether the environment within a Pol III transcriptional domain allowed expression of pre-mRNA. To test this we inserted a CAT gene cassette, seemingly promoterless but endowed with pre-mRNA processing signals, in the chromosome between the U3 snRNA and 7SL RNA genes. Interestingly, abundant CAT mRNA was produced suggesting that the Pol III genes in the immediate vicinity did not prevent access of presumably Pol II to the CAT gene cassette. We propose that either CAT mRNA is synthesized by Pol II run-through transcription or by Pol II initiationupstream from the CAT gene.