The ribosome is the site of protein synthesis in all organisms, and a detailed understanding of ribosome structure is a necessary component of any explanation of the molecular interactions of protein formation. Immunoelectron microscopy is an attractive and useful approach to the localization of ribosomal proteins within the three-dimensional structure of each subunit, but many conflicts have arisen through the use of this technique. A major problem has been the difficulty of purification of ribosomal proteins; highly immunogenic contaminants induce antibody populations directed against proteins other than the one of interest, and immunoelectron microscopy shows incorrect binding sites or multiple distant sites for a single protein. A different approach to immunoelectron microscopic localization of ribosomal proteins is suggested. Ribosomal protein will be rigorously purified by high performance chromatography. Each protein will be chemically derivatized with e.g. DNP, and the modified protein purified by HPLC. Ribosomal subunits will then be reconstituted using a single modified protein plus RNA and all other proteins from control particles. Antibodies directed against the derivatizing reagent, used as a hapten, will then be used to localize the single modified protein by immunoelectron microscopy. Repetition of the experiments with each ribosomal protein will allow their placement using a single well-characterized antibody preparation. It is expected that it will be possible to unambiguously localize all surface ribosomal proteins of each subunit of the Escherichia coli ribosome. In addition, assays of reconstituted-subunit activity should contribute to our understanding of functional neighborhoods and their ribosomal components.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032769-02
Application #
3281877
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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