The ribosome is the physical entity upon which all proteins are biosynthesized. It is both a central point for the regulation of cell growth and a target for external toxins and antibiotics. A detailed understanding of its structure and the relationship between structural components and biological functions in protein biosynthesis is a necessary element in any explanation of the process of translation and its regulation. This proposal details a systematic approach designed to map most or all of the ribosomal proteins within each subunit of the Escherichia coli ribosome. Proteins will be rigorously purified by a combination of reversed phase and ion exchange high performance liquid chromatography. Individual pure proteins will be modified at a small number of sites with fluorodinitrobenzene, and the resulting dinitrophenyl into ribosomal subunits with all other components unmodified proteins will be reconstituted into ribosomal subunits with all others components unmodified, and the reconstituted particles will be characterize physically and functionally. Incorporation of the modified protein will be quantitated, and the site-specificity of its incorporation will be verified. Antibodies to the DNP hapten will then be allowed to react with the subunit, and the immune complexes will be observed by electron microscopy. Localization of each surface protein on each subunit will be established from evaluation of the point of contact of anti-DNP antibody with the ribosomal subparticle. In some cases more than one locus may be established for segments a given protein. If this occurs, attempts will be made to correlate chemical derivitization of specific amino acid residues of the protein with localizations of the protein on the subunit. It is expected that a reliable three dimensional placement of all ribosomal proteins that possess at least one surface-located reactive amino acid residue should be possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032769-08
Application #
2176727
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-04-01
Project End
1994-11-30
Budget Start
1992-12-01
Budget End
1994-11-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Olah, T V; Perrault, A R; Cooperman, B S et al. (1993) Incorporation of dinitrophenyl derivatives of proteins S6, S13, S16, and S18 into the 30 S subunit of Escherichia coli ribosomes by total reconstitution. J Biol Chem 268:18696-700
Montesano-Roditis, L; McWilliams, R; Glitz, D G et al. (1993) Placement of dinitrophenyl-modified ribosomal proteins in totally reconstituted Escherichia coli 30 S subunits. Localization of proteins S6, S13, S16, and S18 by immune electron microscopy. J Biol Chem 268:18701-9
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