Transcription of protein-coding genes in Leishmania

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DESCRIPTION (provided by the applicant): Protein-coding genes in Leishmania and other related Trypanosomatidae are organized and transcribed in a unique fashion, in that they are transcribed polycistronically from large clusters (50-500 kb) of adjacent genes all on the same DNA strand, and the mature mRNAs are generated from the primary transcripts by trans-splicing. Moreover, while this transcription is carried out by RNA polymerase II (pol II), it appears to lack the regulation seen in other organisms, and episomal molecules are transcribed promiscuously. This has led to the hypothesis that pol II has very low specificity in these parasites, and that transcription can initiate indiscriminately throughout the genome. However, our recent transcriptional analysis of the complete chrl from L. major suggests that this is not the case, and that only a small number of specific pol II promoters are present in the genome. Nevertheless, it does appear that these promoters are significantly different from typical eukaryotic promoters, in that they are bi-directional, lack TATA boxes, and have multiple transcription initiation sites (TISs). In addition, the trypanosomatids appear to lack, or have highly divergent versions of, most of the typical eukaryotic pol II transcription factors. Thus, we hypothesize that pol II transcription of protein-coding genes is fundamentally different in these organisms. The recent explosion in sequence information from the trypanosomatid genome projects provides an excellent opportunity to characterize the transcriptional organization of entire chromosomes and identify the transcriptional machinery. In order to test our hypothesis, we intend to: 1. Characterize the transcriptional organization of several chromosomes of L. major Friedlin (LmjF) by Northern blot and nuclear run-on analyses; 2. Characterize the protein components of pol II transcriptional complexes in LmjF by bioinformatic and affinity purification/mass spectrometric approaches; and 3. Investigate the molecular interactions between the pol II machinery and DNA template using both in vitro and in vivo approaches. These studies are designed to elucidate the molecular processes underlying pol II transcription of protein-coding genes in LmjF. We expect that they will reveal fundamental differences between the transcriptional processes of trypanosomatids and other eukaryotes, which can hopefully be exploited for the development of novel chemotherapeutic agents.