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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029362-14
Application #
2175487
Study Section
Molecular Biology Study Section (MBY)
Project Start
1981-09-01
Project End
1998-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
14
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Cornell University
Department
Genetics
Type
Schools of Arts and Sciences
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
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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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