Ribosome Structure and Function

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Project Summary We are entering a new and exciting era of ribosome research, in which the role of ribosome dynamics in protein synthesis is beginning to emerge. This proposal focuses on the role of the structural dynamics of the ribosome, and ribosomal RNA in particular, in the mechanism of translation. Our approach uses a combination of biochemistrty, biophysics, genetics and crystallography. A major goal is to elucidate the mechanism of the coupled translocation of mRNA and tRNA. One strategy is to carry out an exhaustive screen for dominant-lethal mutations in elongation factor EF-G, which we predict will trap the ribosome in previously unobserved transient states of translocation. We then plan to crystallize these trapped complexes and solve their structures, to fill in gaps in our knowledge of the sub-steps of this fundamental process. To link these static, crystallographically-derived snapshots to ribosome dynamics, we will follow internal movements of the ribosome using FRET. We will take advantage of our ability to reconstitute active ribosomes containing fluorescent FRET pairs attached to specific positions, allowing us to follow ribosome dynamics in real time in both ensemble and single-molecule contexts. Mechanistic models derived from these studies can then be tested by creating pure populations of mutant ribosomes using methods that we have previously developed to target mutations to either rRNA or ribosomal proteins, and testing their functional properties in vitro.