The ribosome is the large nucleoprotein complex that uses mRNA as the template and aminoacylated tRNAs as substrates to catalyze protein synthesis in all cells. Ribosomes consist of two subunits in all organisms, designated 30S and 50S in bacteria, which together form the 70S ribosome. The 30S subunit improves the fidelity of translation by monitoring codon-anticodon interactions, while the 50S catalyzes peptide bond formation. Both subunits act in concert during translocation, which involves the movement of mRNA and tRNA through the ribosome. Many clinically important antibiotics target the bacterial ribosome. Recent advances, including those from our own laboratory, have resulted in high resolution structures of each subunit and a medium resolution structure of the whole 70S ribosome. These structures have revolutionized our understanding of ribosome structure and function. This is a proposal to build on these advances. We have elucidated the interactions of several antibiotics with the 30S subunit, and propose to determine the structure of several important remaining ones. We shall also determine the structure of the 30S subunit in complex with initiation factors, protein S1, and with a variety of tRNA and mRNA combinations that involve non-standard pairing or modified bases at the third position of the codon, the wobble position. A special RNA called tmRNA, because it has both tRNA and mRNA-like properties, is used by the cell to rescue ribosomes stalled on defective messages. We shall determine the structure of the ribosome in complex with tmRNA in various states. We shall also crystallize complexes of the ribosome specifically arrested at various points along the translation pathway. Solution of crystal structures of these complexes will shed light on the mechanisms involved during translation, including the interaction of factors with the ribosome and conformational changes during translation. These studies will not only shed light on fundamental aspects of translation, a central process in all cells, but also reveal how many antibiotics interact with the ribosome. Such knowledge could help improve existing antibiotics and could also lead to the design of new ones.
