The overall goal of this project is to understand the control of mitochondrial gene expression in the model eucaryote, yeast. The research will focus on transnational control of the mitochondrial COX2 and COX3 mRNAs.
The specific aims are: 1) Determine the mechanism of mRNA-specific transnational activation. The specific activation of COX3 mRNA translation by a membrane-bound complex containing the nuclearly coded proteins PET54, PET122 and PET494 will be studied further. Additional components involved in translation activation will be identified, genetically and biochemical. The complex will be purified and used to test in vitro for interactions with both the 613 base COX3 mRNA 5'-untranslated leader (UTL) and with mitochondrial ribosomes. Transnational activation of the COX2 mRNA by the PET2111 protein will be studied by identifying components that interact with PET111, and exhaustively analyzing the site in the 54 base COX2 mRNA 5'-UTL where PET111 acts. To determine the spatial requirements for activation in vivo, both the COX2 and COX3 activation sites will be moved from the 5'- UTLs to the mRNA 3'trailers, and dicistronic mRNAs will be constructed. 2) Express a reporter gene and/or selectable marker located in the mitochondrial genome. To allow quantitative measurement of mitochondrial gene expression the nuclear gene ARG8 will be inserted into the mitochondrial genome in such a way that it should be expressed. ARG8 encodes the soluble enzyme acetylornithrine transaminase, which is normally located in the mitochondrial matrix and can be assayed in yeast cell extracts. Expression in mitochondrial translation initiation sites. Yeast mitochondrial initiation sites are specified by both AUG codons and some other context information. Mutational analysis will be used to define initiation sites in vivo. 4) Characterize the responses of mitochondrial gene expression to different environmental and developmental conditions. The levels of COX2 and COX3 expression will be surveyed under a variety of environmental and developmental conditions. The rate limiting factors responsible for modulation will be identified genetically. 5) Determine the extent of conservation among other fungi of the transnational activation system discovered in Saccharomyces. Sequence homologues of Saccharomyces transnational activators in Kluyveromyces lactis and Candida glabrata will be identified and disrupted in those yeasts to determine whether they are functional homologues. The information gained will be used for a sensitive search for homologues in pathogenic fungi.
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