The ribosome, the physical site of protein biosynthesis in all cells, is a central point for control of cell growth and metabolism. It plays a role in the regulation of expression of specific gene products. It is an important target for external toxins and antibiotics; ribosome inactivation blocks protein synthesis. Detailed knowledge of the structure and function of the ribosome is thus a major part of any understanding of the molecular mechanisms of protein biosynthesis and its regulation. This proposal is concerned with the structural characterization of the ribosome, and in particular with mapping the location of the messenger and transfer RNA molecules that are involved in translation and with localizing the elements of the ribosomal RNA that interact with them. A long term aim is mapping the overall folding of the RNA species that make up almost two thirds of the mass of each prokaryotic ribosomal subunit. Oligonucleotides that mimic mRNA or tRNA and short oligonucleotides that complement specific functionally important short segments of ribosomal RNA will be synthesized and their ability to efficiently hybridize to ribosomes or ribosomal subunits will be optimized. Useful oligonucleotide probes will be modified so as to carry either a very small (14 A) gold cluster or an antibody-recognizable derivative and then bound to ribosomes or subunits. Electron microscopy of ribosome-oligonucleotide-gold complexes or antibody-oligonucleotide-ribosome complexes will then allow localization of the oligonucleotide (and thus the mRNA element, tRNA segment, or ribosomal RNA sequence that it complements) on the surface of the ribosome, using the gold cluster or the antibody molecule as a visible pointer. Functional assays will be used to correlate physical sites with individual steps in the protein biosynthetic process. The ultimate aim is a three dimensional map of the ribosome that shows the general conformation of the RNA molecules and explains their function in the structure of the ribosome and in the many interactions and reactions involved in protein biosynthesis.
