Department of Biochemistry and Molecular Biology
College of Medicine, Health Science Center
University of Florida
ainesville,
Florida 32610-0245
Phone: (352) 392-6878
FAX: (352) 392-2953
Research Focus | CurrentResearch | Selected Publications |Research Presentations |Surprises from Mitoribosome Research
Mammalian mitochondrial ribosomes differ unexpectedly from bacterial and cytoplasmic ribosomes, as well as other kinds of mitochondrial ribosomes.
The bovine mitochondrial ribosome is being developed as a model system for the study of mammalian mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Current projects include structural/functional characterization of the novel GTP binding site on mitochondrial ribosomes, initiation factors and mRNA binding to mitochondrial ribosomes, a structural model for mitochondrial ribosomes, and the structure, organization and expression of genes for mitochondrial ribosomal proteins.
A. Wesley H. Faunce and Tom O'Brien:
3D model of bovine mitoribosomal small subribosomal particle (small ubunit), using coordinates (Harry Noller Lab) of conserved nucleotides, coordinates of conserved proteins (Peter Moore Lab) and electron microscopic imaging of bovine 28S subunits (Tom and Bonnie O'Brien). Model being updated and refined, using coordinates of conserved nucleotides and proteins from the recent (2000, 2001) crystallographic structures determined for ribosomal subunits of Thermus thermophilus .
B. Earlier work, by Russell J. Salfi and Tom O'Brien:
Tom O'Brien, working with Russell J. Salfi, UF TRUE fellow '98, developed a 3D computer rendering of the tertiary structure of the mammalian (bovine) mitochondrial small ribosomal subunit in order to gain understanding of the structure and function of this protein rich ribosome. Renderings, freeware programs, and TI-83 programs used to generate these renderings can be found at: http://www.med.ufl.edu/biochem/tobrien/3dmodel/
C. Robert Stiff and Tom O'Brien:
Hybrid structural model development incorporating information on the chemical and enzymatic accessibility of mitoribosomal RNA in the 3D model of the bovine 28S subunit.
2. Genes for mammalian mitoribosomal proteins
A. Jiguo Liu and Tom O'Brien:
(1) Cloning and analysis of cDNA and genes for the human (MRPL12) and bovine (MRPL31, in bovine nomenclature) mitoribosomal proteins homologous to the eubacterial ribosomal protein L7/L12.
(2) Cloning and analysis of cDNA and genes for the human and bovine mitoribosomal protein S5 (MRPS29, HGNC nomenclature), the GTP binding protein in the small subunit of mammalian mitoribosomes.
B. Scott Fiesler and Tom O'Brien:
Cloning and analysis of cDNA for bovine mitoribosomal protein L2 (MRPL37, HGNC nomenclature), the target of chloramphenicol affinity analogues in the large subunit of mammalian mitoribosomes (no homologue in bacterial or cytoplasmic ribosomes!).
C. Hanns-Ruediger Graack, Brigitte Wittmann-Liebold and Tom O'Brien:
Analysis and evolution of mammalian ribosomal proteins
3. CONSORTIUM to Clone and Map and Study Human Mitochondrial Ribosomal Protein Genes
In addition to my laboratory, the following laboratories (listed alphabetically) are participating in this consortium:
Nathan Fischel-Ghodsian, Cedars-Sinai Medical Center, Los Angeles, CA
Hanns-Ruediger Graack, Institute for Genetics, AG Kress, Free University of Berlin, Germany
James E. Sylvester, Nemours Children's Clinic, Jacksonville, FL
Brigitte Wittmann-Liebold, Max-Delbruck-Center for Molecular Medicine, Berlin, Germany
Mammalian mitochondria have their own separate translation system comprised of components distinct from their cytoplasmic counterpart. Whereas mitochondrial DNA encodes tRNAs and the two rRNAs, the remaining genes for the translation system are found in nuclear DNA. Upward of 100 mitochondrial ribosomal proteins (MRPs) are imported into the mitochondria, assembled into ribosomes which are responsible for translating the 13 mRNAs for oxidative phosphorylation proteins. Since mutations in mitochondrial tRNA and rRNA can cause various pathological states, we hypothesize that mutations in MRP genes are also candidates for human disorders. A necessary first step, therefore, is to identify, clone, and map the gene for each human MRP. Our approach is to use N-terminal and internal amino acid sequence data obtained from purified bovine or rat MRPs (Goldschmidt-Reisin, et al., J.Biol.Chem. 273: 34828, 1998) to search EST databases. A representative I.M.A.G.E. clone (ATTC) is purchased and used to screen a human lambda genomic library. Chromosome map positions are ascertained by in situ hybridization (FISH) with genomic sequences and/or by using in silico methods to search Genbank and GeneMap through NCBI. At present, we have over 30 different human MRPs at various stages of characterization and have established a consortium of investigators to complete the project. We are currently investigating one MRP as a potential candidate for Russell-Silver Syndrome (RSS). In addition to studying their clinical relevance, long term characterization of MRP genes should lead to important insights into mammalian evolution, coordinate regulation of nuclear and mitochondrial gene expression, and ribosome function.
4. GTP binding site on bovine mitoribosomal small subunit
John Anders, Jiguo Liu, Nancy Denslow and Tom O'Brien
5. Mitoribosomal mRNA binding site
Bernard Courtney, Robert Heck, Nancy Denslow and Tom O'Brien
O'Brien TW, Liu J, Sylvester JE, Mougey EB, Fischel-Ghodsian N, Thiede B, Wittmann-Liebold B, Graack HR.Mammalian Mitochondrial Ribosomal Proteins (4): Amino Acid Sequencing, Characterization, and Identification of Corresponding Gene Sequences. J Biol Chem. 275: 18153-18159 (2000).
Spirina, O., Bykhovskaya, Y., Kajava, A.V., O'Brien, T.W, . Nierlich, D.P., Mougey, E.B., Sylvester, J.E., Graack,, H.-R., Wittmann-Liebold, B. and Fischel-Ghodsian N. Heart-specific splice-variant of a human mitochondrial ribosomal protein, Gene, 261:229-234. (2000)
Samaha, R.S., Joseph, S., O'Brien, B., O'Brien, T.W. and Noller, H.F. Site-directed hydroxy radical probing of 30S ribosomal subunits by using Fe(II) tethered to an interruption in the 16S rRNA chain. Proc. Natl. Acad. Sci.USA., 96: 366-370 (1999).
O'Brien, T.W., and Denslow, N.D., Bovine Mitochondrial Ribosomes, In: Methods in Enzymology: Mitochondrial Genetics and Biogenesis, 264:237-248 (1996).
Samaha, R.S., O'Brien, B., O'Brien, T.W.and Noller, H.F. Independent In Vitro Assembly of a Ribonucleoprotein Particle Containing the 3' Domain of 16S rRNA. Proc. Natl. Acad.Sci.USA., 91: 7884-7888 (1994).
Aroor, A.R., Denslow, N.D., Singh, L.P.,O'Brien, T.W. and Wahba, A.J. Phosphorylation of the Rabbit Reticulocyte Guanine Nucleotide Exchange Factor In Vivo. Identification of Putative Casein Kinase II Phosphorylation Sites. Biochemistry, 33: 3350-3357 (1994).
O'Brien, T.W., Denslow, N.D., Faunce, W.H.,Anders, J.C., Liu, J., and O'Brien, B.J., Structure and Function ofMammalian Mitochondrial Ribosomes, In: The Translational Apparatus: Structure, Function, Regulation, Evolution. Nierhaus, K.H., Franceschi, F., Subramanian, A.R., Erdmann, V.A. and Wittman-Liebold, B. (eds.) Plenum Publishing Corp., New York (1993) pp. 575-586.
Pietromonaco, S.F., Denslow, N.D. and O'Brien, T.W., Proteins of Mammalian Mitochondrial Ribosomes. Biochimie, 73:827-836 (1991).
Denslow, N.D., Anders, J.C., and O'Brien,T.W., Bovine Mitochondrial Ribosomes Possess a High Affinity Binding Site for Guanine Nucleotides, J. Biol. Chem., 266:9586-9590(1991).
O'Brien, T.W., Denslow, N.D., Anders, J.C.,and Courtney, B.C., The Translation System of Mammalian Mitochondria, Biochim. Biophys. Acta, 1050:174-178 (1990).
O'Brien, T.W., Denslow, N.D., Anders, J.C.,Attardi, G. and O'Brien, B.J., Role of Initiation Factors and Ribosomal Proteins in Mammalian Mitochondrial Gene Expression, In: Structure, Function and Biogenesis of Energy Transfer Systems, E. Quagliariello, S. Papa, F. Palmieri, C. Saccone, (eds.), Elsevier, (1990) pp. 153-158.
Denslow, N.D., O'Brien, T.W., Michaels,G.S., Montoya, J. and Attardi, G., Mechanism of mRNA Binding to Bovine Mitochondrial Ribosomes, J. Biol. Chem., 264: 8328-8338 (1989).
Piatyszek, M., Denslow, N.D. and O'Brien,T.W., RNA Binding Proteins of the Large Subunit of Bovine Mitochondrial Ribosomes, Nucleic Acid Research, 16: 2565-2583 (1988).
Denslow, N.D., LiCata, V.J., Gualerzi, C.and O'Brien, T.W., The Interaction of Bovine Mitochondrial Ribosomes with E. coli Initiation Factor 3 (IF3). Biochemistry, 27: 3521-3527 (1988).
Chiles, T.C., O'Brien, T.W. and Kilberg,M.S., Production of Mono-Specific Antibodies to a Low-Abundance Hepatic Membrane Protein Using Nitrocellulose Immobilized Protein as Antigen. Anal. Biochem., 163: 136-142 (1987).
Pietromonaco, S., Hessler, R.A. and O'Brien, T.W., Evolution of Proteins in Mammalian Cytoplasmic and Mitochondrial Ribosomes. J. Mol. Evolution, 24:110-117 (1986).
O'Brien, T.W., Pietromonaco, S.F.,Piatyszek, M.A. and Gillevet, P.M., Genes for Mammalian Mitoribosomal Proteins. In: Achievements and Perspectives in Mitochondrial Research, Vol. II, E. Quagliariello, E.C. Slater, F.Palmieri, C. Saccone, and A.M. Kroon (eds.), Elsevier/North Holland, (1985) pp. 379-387.
Schieber, G.L. and O'Brien, T.W., Site of Synthesis of the Proteins of Mammalian Mitochondrial Ribosomes: Evidence from Cultured Bovine Cells. J. Biol. Chem., 260:6367-6372 (1985).
Denslow, N.D. and O'Brien, T.W.,Organization of Proteins in Mammalian Mitochondrial Ribosomes: Accessibility to Lactoperoxidase-catalyzed radio-iodination. J.Biol. Chem., 259:9867-9873 (1984).
Schieber, G.L. and O'Brien, T.W.,Cytoplasmic Synthesis of Proteins of the Mammalian Mitochondrial Ribosome. In: Nucleo-Mitochondrial Interactions, R. Schweyenand F. Kaudewitz (eds.), De Gruyter Press (1983), pp.469-479.
Schieber, G.L. and O'Brien, T.W., Extraction of Proteins from the Large Subunit of Bovine Mitochondrial Ribosomes under Nondenaturing Conditions. J. Biol. Chem., 257:8781-8787 (1982).
Matthews, D.E., Hessler, R.A., Denslow,N.D., Edwards, J. and O'Brien, T.W., Protein Composition of Bovine Mitochondrial Ribosomes. J. Biol. Chem., 257: 8788-8794(1982).
O'Brien, T.W., Denslow, N.D., Harville,T.O., Hessler, R.A. and Matthews, D.E., Functional and Structural Roles of Proteins in Mammalian Mitochondrial Ribosomes. In: The Organization and Expression of the Mitochondrial Genome, A. Kroon and C. Saccone (eds.), Elsevier/North-Holland Biomedical Press (1980) pp. 301-305.
Hauswirth, W.W., Laipis, P.J., Gilman,M.E., O'Brien, T.W., Michaels, G.S. and Rayfield, M.A., Genetic Mapping of Bovine Mitochondrial DNA from a Single Animal. Gene, 8:193-209 (1980).
Laipis, P.J., Hauswirth, W.W., O'Brien,T.W. and Michaels, G.S., A Physical Map of Bovine Mitochondrial DNA from a Single Animal. Biochem. Biophys. Acta, 565:22-32 (1979).
Denslow, N.D., and O'Brien, T.W.,Elongation Factors EF-G from E. coli and Mammalian Mitochondria are not functionally interchangeable. Biochem. Biophys. Res.Commun., 90:1257-1265 (1979).
Denslow, N.D. and O'Brien, T.W., Antibiotic Susceptibility of the Peptidyl Transferase Locus of Bovine Mitochondrial Ribosomes. Eur. J. Biochem., 91:444-448(1978).
Matthews, D.E., Hessler, R.A. and O'Brien,T.W., Rapid Evolutionary Divergence of Proteins in Mammalian Mitochondrial Ribosomes. FEBS Letters, 86:76(1978).
O'Brien, T.W., Transcription and Translation in Mitochondria. In: International Cell Biology,1976-77, B.R. Brinkley and Keith R. Porter (eds.), The Rockefeller University Press, New York (1977) pp. 245-255.
O'Brien, T.W., Matthews, D.E. and Denslow, N.D., Protein Composition of the Bovine Mitochondrial Ribosome. In: The Genetics and Biogenesis of Chloroplasts and Mitochondria, T. Bucher, W. Neupert, W. Sebald and S. Werner (eds.), North-Holland Publishing Co., Amsterdam (1977) pp. 741-748.
O'Brien, T.W., and Matthews, D.E., Mitochondrial Ribosomes. In: Handbook of Genetics, Vol. 5, Robert C. King (ed.), Plenum Publishing Co., 1976, pp. 535-80.
O'Brien, T.W., Mitochondrial Protein Synthesis. In: Protein Synthesis, Vol. 2, E.H. McConkey (ed.), Marcel Dekker, Inc., New York, 1976, pp. 249-307.
Hamilton, M.G. and O'Brien, T.W.,Ultracentrifugal Characterization of the Mitochondrial Ribosome and Subribosomal Particles of Bovine Liver: Molecular Size and Composition. Biochemistry, 13:5400 (1974).
Denslow, N.D. and O'Brien, T.W.,Susceptibility of 55S Mitochondrial Ribosomes to Antibiotics Inhibitory to Prokaryotic Ribosomes: Lincomycin, Chloramphenicol, and PA114A. Biochem. Biophys. Res. Commun., 57:9 (1974).
O'Brien, T.W., The General Occurrence of 55S Ribosomes in Mammalian Liver Mitochondria. J. Biol. Chem.,245:3409 (1971).
O'Brien, T.W. and Kalf, G.F., Ribosomes from Rat Liver Mitochondria. II. Partial Characterization. J. Biol. Chem., 242:2180 (1967).
O'Brien, T.W. and Kalf, G.F., Ribosomes from Rat Liver Mitochondria. I. Isolation Procedure and Contamination Studies. J. Biol. Chem., 242:2172 (1967).
ResearchPresentations:
Liu, J. and O'Brien, T.W. Structural Disposition of Mammalian Mitoribosomal Proteins: Bovine MRP-L31, a Member of the Eubacterial L7/L12 Superfamily. International Conference on the Ribosome: Structure, Function, Antibiotics and Cellular Interactions, Helsingoer, Denmark, June 13 - 17, 1999.
Graack, H.-R., O'Brien, T. W., and Wittmann-Liebold, B. Mitochondrial Ribosomal Proteins (MRPs) from Yeast and Mammals: Identification, Characterization, and Evolution. 23rd Annual meeting of the German Cell Biology Society. Rostock, March 14-18, 1999; Eur. J. Cell Biol. (1999) 78 suppl. 49, 91
Liu, J., Barnoski B. L., and O'Brien T. W. Structure and organization of human genes for mitoribosomal proteins: the homologue of the prokaryotic ribosomal protein L7/L12. Annual Meeting of the American Society for Human Genetics, Denver, October 27 - 31, 1998.
O'Brien, T. W., Faunce, W. and Cogle,C. RNA Domains in Mammalian Mitochondrial Ribosomes, Third Annual Meeting of the RNA Society, Madison, May 26-31, 1998.
Liu, J. and O'Brien, T. W. Characterizationof Genomic DNA for Bovine Mitoribosomal Protein L31, a Member of the Eubacterial L7/L12 Superfamily, Annual Meeting of the American Society for Biochemistry and Molecular Biology, Washington, D.C., May 16-20, 1998.
Liu, J. and O'Brien, T. W. Cloning of cDNAand Genomic DNA for Bovine Mitoribosomal Protein L31, a Member of the Eubacterial L7/L12 Superfamily, 17th International Congress of Biochemistry and Molecular Biology, San Francisco, August 24-29, 1997.
O'Brien, T., Faunce, W., Cogle, C. and Heck, R. RNA Domains in Mammalian Mitochondrial Ribosomes, RNA Structure Symposium, University of California, Santa Cruz, June 25-26, 1997.
O'Brien, T., Faunce, W. and Cogle, C. RNA Disposition in Mammalian Mitochondrial Ribosomes. Second Annual Meeting of the RNA Society, Banff, Alberta, Canada, May 27-June 1, 1997.
Liu, J. and O'Brien, T.W. Cloning of cDNA for Bovine Mitoribosomal Protein L31, A Member of the Eubacterial L7/12 Superfamily. International Congress on Cell Biology, SanFrancisco, California, December 7-11, 1996.
Liu, J., Anders, J. C. and O'Brien, T. W.GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes: a Protein in Search of Function. Cold Spring Harbor Meeting on Translational Control, September 4 - 8, 1996.
Samaha, R., Joseph, S., O'Brien, B.,O'Brien, T. W. and Noller, H.F. Site-Directed Hydroxyl Radical Probing Using 30S Ribosomal Subunits Assembled in vitro from Synthetic 16S rRNA Fragments. The First Annual Meeting of the RNA Society, Madison, Wisconsin, May 28-June 2, 1996.
Liu, J., Bryant, M. and O'Brien, T.W.Purification, Sequence Analysis and Cloning of cDNA for Bovine Mitoribosomal Proteins L31, Superfamily. The First Annual Meeting ofthe RNA Society, Madison, Wisconsin, May 28-June 2, 1996.
Liu, J. and O'Brien, T.W. Cloning of cDNA for Bovine Mitoribosomal Protein L31, A Member of the Eubacterial L7/12 Superfamily. Gordon Research Conference on Mitochondria and Chloroplasts, June 16-21, 1996.
O'Brien, T. W., Faunce, W. H., O'Brien, B.J., Cogle, C.R. and Hussain, S. RNA Domains in Mitochondrial Ribosomes: Disposition of RNA in the Small Subunit of Bovine Mitochondrial Ribosomes. The First Annual Meeting of the RNA Society, Madison, Wisconsin May 28-June 2, 1996.
Liu, J., Anders, J.C., and O'Brien, T.W. Purification, Sequence Analysis and Cloning of cDNA for the GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes, Ninth Symposium of the Protein Society, Boston, July 8-12, 1995.
Liu, J., Anders, J.C., and O'Brien,T.W. Purification, Sequence Analysis and Cloning of cDNA fort he GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes, International Conference on the Structure and Function of the Ribosome, "Frontiers in Translation", Victoria, B.C., May 20-25, 1995.
O'Brien, T.W., Faunce, W.H., O'Brien, B.,Cogle, C.R, and Hussain, S. RNA Domains in Mitochondrial Ribosomes: Disposition of RNA in the Small Subunit of Bovine Mitochondrial Ribosomes, International Conference on the Structure and Function of the Ribosome, "Frontiers in Translation", Victoria, B.C., May 20-25, 1995.
O'Brien, T.W., Liu, J., and Anders,J.C. Sequence and Properties of the GTP Binding Protein in Mammalian Mitochondrial Ribosomes, International Symposium on Thirty Years of Progress in Mitochondrial Bioenergetics and Molecular Biology, Selva di Fasano, Italy, October 7-10, 1994.
O'Brien, T.W., Faunce, W.H., O'Brien, B.J.,and Cogle, C.R. RNA Domains in Mitochondrial Ribosomes; Disposition of RNA in the small subunit of Bovine Mitochondrial Ribosomes, International Symposium on Thirty Years of Progress in Mitochondrial Bioenergetics and Molecular Biology, Selva di Fasano, Italy, October 7-10, 1994.
Liu, J., Anders, J.C., and O'Brien, T.W.Amino Acid Sequence Analysis and Cloning of cDNA for the GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes, Cold Spring Harbor Meeting on Translational Control, August 24-28,1994.
Liu, J., Anders, J.C., and O'Brien, T.W. Purification, Sequence Analysis and Cloning of cDNA for the GTP Binding Protein in the small subunit of Bovine Mitochondrial Ribosomes, Abstract LBl9, American Society for Biochemistry and Molecular Biology Meeting, Washington, D.C., May 21-25, 1994.
O'Brien, T.W., Faunce, W.H., O'Brien, B.J.,and Cogle, C.R. RNA Domains in Mitochondrial Ribosomes; Disposition of RNA in the small subunit of Bovine Mitochondrial Ribosomes, Gordon Research Conference on Mitochondrial and Chloroplasts, Volterra, Italy, May 1-6, 1994.
Courtney, B.C. and O'Brien, T.W., Binding of Mitochondrial mRNA to Bovine Mitochondrial Ribosomes, Gordon Research Conference on Mitochondria and Chloroplasts, Volterra, Italy, May 1-6, 1994.
O'Brien, T.W., Faunce, W.H., Courtney,B.C., Anders, J., Liu, J., and Heck, R., Structure and Function of Mammalian Mitochondrial Ribosomes, International Conference on theTranslational Apparatus, Berlin, October 31-November 5, 1992.
Denslow, N.D., Arroor, A.R., O'Brien, T.W.,and Wahba, A.J., In Vitro and In Vivo Phosphorylation of the Guanine Nucleotide Exchange Factor, Cold Spring Harbor, New York, September9-13, 1992.
Faunce, W. and O'Brien, T.W., Chemical and Enzymatic Probing of the 12S RNA Structure in the Small Subunit of Bovine Mitochondrial Ribosomes, The FASEB J., 4: A2002, (1990).
Denslow, N.D., Anders, J.C., Courtney,B.C., Sonnega, C.E.A. and O'Brien, T.W., Role of Initiation Factors and Ribosomal Proteins in Mammalian Mitochondrial Gene Expression, Cold Spring Harbor Symposium on Translational Control, Cold Spring Harbor, New York, September 13-17, 1989.
Denslow, N.D., Anders, J.C., Sonnega, C.E.A. and O'Brien, T.W., Characterization of a Novel GTP Binding Site in Mammalian Mitochondrial Ribosomes, American Society for Microbiology Conference on Ribosomes, East Glacier Park, Montana,August 6-11, 1989.
O'Brien, T.W., Anders, J.C., and Denslow, N.D., Purification and Microsequence Analysis of a GTP-binding Protein from Mammalian Mitochondrial Ribosomes, American Society for Microbiology Conference on Ribosomes, East Glacier Park, Montana,August 6-11, 1989.
O'Brien, T.W., Anders, J.C., Denslow, N.D.and Pietromonaco, S., Distinctive Properties of 55S Animal Mitochondrial Ribosomes, American Society for Microbiology Conference on Ribosomes, East Glacier Park, Montana, August 6-11, 1989.
