The bacterial ribosome is a complex organelle composed of 3 RNA molecules and 52 proteins. In order to understand the molecular mechanisms of protein synthesis, the faithful translation of genetic information, it will be necessary to determine the three-dimensional structure of both the small and large ribosome subunits. We shall prepare a library of monoclonal antibodies to each of the proteins of the ribosome subunits. We will use these antibodies to map the locations of antigenic determinants of the ribosomal proteins on the ribosome surface by immunoelectron microscopy. Proteins which are not exposed to anitbody on the ribosome surface will be located on protein dificient ribosome subparticles prepared by high salt extraction of subunits or by reconstitution from purified components. We shall detect possible protein interactions within the ribosome subunits by inhibition of antibody binding as the subunits are assembled in vitro. The availability of highly specific monoclonal antibodies to all of the ribosomal proteins will facilitate the localization of functional sites by affinity labelling and the identification of RNA-protein interactions within the subunits by photochemical crosslinking. The location of functional sites with the ribosome structure should further our understanding of the molecular mechanisms of initiation, regulation of translational fidelity, translocation and termination and other fundamental processes necessary for the translation of genetic information into functional proteins.
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| Syu, W J; Kahan, L (1987) Use of protein-stained immunoblots for unequivocal identification of antibody specificities. J Immunol Methods 103:247-52 |
