TRANSLATION SYSTEM IN THE KINETOPLAST OF LEISHMANIA

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A goal of the proposed research is to verify existence of a functional system of translation in the kinetoplast-mitochondrion of Leishmania and investigate its basic properties. This is necessary for understanding the kinetoplast functions in the parasite's life cycle. Identification of the components of the translation machinery may provide useful therapeutical targets for disrupting the life cycle and treatment of trypanosomatid-caused diseases. In addition, since translation operates with templates produced by RNA editing, these two systems may interact in a way that has no precedent in other eukaryotic systems. Studies of kinetoplast ribosomes and translation regulatory factors may uncover novel regulatory mechanisms and structural principles. Specific aims are as follows: #1. Detection and characterization of proteins derived from fully edited mRNA. This will be investigated with antibodies against several mitochondrial proteins expressed in heterologous system or synthetic peptides. Regulation on translational level will be studied by analysis of edited mRNA and protein levels throughout the Leishmania life cycle. #2. Characterization of a translation system in the kinetoplast. To investigate de novo protein synthesis, incorporation of radioactive amino acids into high molecular weight products in kinetoplasts- mitochondria will be studied using intact cells and isolated kinetoplast-mitochondrial fraction. Changes in the pattern of de novo synthesized proteins during the life cycle will be investigated. #3. Identification and isolation of kinetoplast ribosomes. Ribosomes will be identified with specific probes for 9S and 12S. rRNA and antibodies against ribosomal protein S12. Functional evaluation of putative ribosomes will be performed by assaying their translation competence in vivo and in vitro. Attempts will be made to reconstitute a cell-free system of kinetoplast translation using homologous extract and purified ribosomes. Physico-chemical properties and composition of the kinetoplast ribosomes will also be studied. #4. Identification and isolation of mRNA binding proteins. In order to investigate mechanisms which prevent initiation of translation on immature mRNA, proteins which specifically recognize edited or pre- edited mRNA will be identified.