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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029362-09
Application #
3276950
Study Section
Genetics Study Section (GEN)
Project Start
1981-09-01
Project End
1994-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Earth Sciences/Resources
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Elliott, Leah E; Saracco, Scott A; Fox, Thomas D (2012) Multiple roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase. Genetics 190:559-67
Fox, Thomas D (2012) Mitochondrial protein synthesis, import, and assembly. Genetics 192:1203-34
Kuhl, Inge; Fox, Thomas D; Bonnefoy, Nathalie (2012) Schizosaccharomyces pombe homologs of the Saccharomyces cerevisiae mitochondrial proteins Cbp6 and Mss51 function at a post-translational step of respiratory complex biogenesis. Mitochondrion 12:381-90
Mick, David U; Fox, Thomas D; Rehling, Peter (2011) Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation. Nat Rev Mol Cell Biol 12:14-20
Yogev, Ohad; Yogev, Orli; Singer, Esti et al. (2010) Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response. PLoS Biol 8:e1000328
Shingu-Vazquez, Miguel; Camacho-Villasana, Yolanda; Sandoval-Romero, Luisa et al. (2010) The carboxyl-terminal end of Cox1 is required for feedback assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria. J Biol Chem 285:34382-9
Bonnefoy, Nathalie; Fiumera, Heather L; Dujardin, Geneviève et al. (2009) Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes. Biochim Biophys Acta 1793:60-70
Fiumera, Heather L; Dunham, Maitreya J; Saracco, Scott A et al. (2009) Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1 in the absence of Cox18. Genetics 182:519-28
Perez-Martinez, Xochitl; Butler, Christine A; Shingu-Vazquez, Miguel et al. (2009) Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria. Mol Biol Cell 20:4371-80
Ding, Martina G; Butler, Christine A; Saracco, Scott A et al. (2008) Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins. Biochim Biophys Acta 1777:1147-56

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