Atomic-resolution structure and function of the ribosome

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DESCRIPTION (provided by applicant): The ribosome plays a key role in the conversion of genotype into phenotype in all forms of life. It is the large RNA and protein factory responsible for protein synthesis, and translates the genetic code in messenger RNA into the corresponding protein. Recent biochemical and structural studies of the ribosome have started to reveal the molecular basis of translation. Images of the two ribosomal subunits have been determined individually at atomic resolution by x-ray crystallography. However, the individual ribosomal subunits lack many functions of the ribosome necessary for protein synthesis. The ribosomal subunits must work together as an intact ribosome, in order for translation to occur. In order to provide a complete picture of the protein synthesis cycle at the molecular level, an atomic resolution "movie" of the intact ribosome, adding an amino acid to a growing polypeptide chain, will be necessary. We propose to make the first "frames" of this movie, by determining the atomic-resolution structures of two intact ribosomes from the model organism, Escherichia coli. By focusing on the E. coli ribosome, we will be able to compare our structural results directly to decades of biochemical and genetic research on translation, that has been carried out with this model system. During the first four years of this grant, we determined x-ray crystal structures of the entire  coli ribosome at resolutions of 9-12 A, that shed some light on the structural basis of translation. These structures provide a first step in our goal of making "snapshots" of the ribosome at atomic resolution during the protein elongation cycle. We have now obtained crystals of the intact E. coli ribosome that diffract x-rays to atomic resolution. These crystals will allow us, for the first time, to image the ribosome at atomic resolution, and to explore, in atomic detail, the impact of ribosomal mutations that confer antibiotic resistance. Our results will significantly impact our understanding of translation, and will be widely useful in many biological fields where translation plays an important role. The specific aims of the proposal are as follows: 1. Solve and refine atomic-resolution structures of two intact 70S ribosomes from E. coli. 2. Determine the functional states of the two  coli ribosome structures. 3. Probe the structural and functional consequences of mutations in the ribosome that cause antibiotic resistance.