In all eucaryotes, the expression of mitochondrial genes within the organelle must be coordinated with the expression of nuclear genes. The long term goal of this project is to understand the molecular mechanisms by which mitochondrial gene expression is regulated. This proposal seeks continued support for the study of nuclear genes that specifically control expression of the Saccharomyces cerevisiae mitochondrial genes encoding cytochrome oxidase subunits II (coxII) and III (coxIII). The nuclear genes under study have been shown, during the previous grant period, to activate translation of the mitochondrially coded mRNAs.
The specific aims are: 1) Determine the mechanisms by which translation o the mitochondrially coded mRNAs for coxIII and coxIII is activated by nuclear genes. The key goal here is to identify the target molecules with which the known positive regulatory proteins interact. The action of the nuclear genes PET494, PET54 and PET122 on coxIII translation will be study by: a) Precisely mapping the site(s) of action for the nuclear genes on the 5'- leader of the coxIII mRNA, using a new procedure for mitochondrial transformation to study the in vivo effect of in vitro-generated mutations. b) Selecting second site suppressors of mutations in the coxIII mRNA 5'-leader and in the three nuclear genes. c) Purifying the products of the three nuclear genes, to allow in vitro studies of protein-RNA binding and to determien as precisely as possible their intra-mitochondrial location (are they, for example, associated with ribosomes?). the action of PET111 on coxII translation will be studied by determining whether PET111 acts in the coxII mRNA 5'-leader to promote translation, or whether it acts by a more complex mechanism involving the N-terminus of the coxII-precursor protein. Second site suppressors of pet111 mutations will be selected. 2) Determine how the nuclear genes PET494, PET54, PET122 and PET111 are regulated. The primary goal here is to begin to understand what role these nuclear may have in modulating mitochondrial gene expression. However, their regulation is also intrinsically interesting, since at least one of them, PET494, is post-transcriptionally controlled. 3) Determine whether mitochondrial ribosomes scan of AUG codons. The goal here is to study general features of mitochondrial translation initiation. AUG codons and stable RNA stems will be placed in the 5'-leader of the coxII mRNA, and the normal AUG initiation codon will be mutated. Any mutations that affect translation will be used to select revertants and pseudorevertants.
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