Molecular analysis of tRNA-mRNA movement in the ribosome

Ribosomes are megadalton ribonucleoprotein particles dedicated to protein synthesis. The process of protein synthesis, also called translation, is one of the major targets of antibiotics that inhibit bacterial cell growth. During translation, the ribosome interactively orchestrates 3 major events: decoding, peptidyl transfer, and translocation. Translocation, coupled movement of tRNA and mRNA in the ribosome, is catalyzed by elongation factor G (EF-G).In Chapter 1, I introduce the molecular mechanisms of translocation in light of both previous and concurrent studies after the 40 years of research of this field. Translocation involves movement of tRNA and mRNA to their adjacent sites some &sim40 A away at the longest distance. The movement occurs in a step-wise manner with a series of conformational changes both in the ribosome and EF-G. Biochemical studies have elucidated the kinetics of translocation and how ribosomes control the precise movement of tRNA and mRNA. I also cover the recent advance in X-ray crystallography and cryoelectron microscopy that have provided us with an unprecedented amount of information on structures of ribosomal complexes during translocation.Chapter 2 is focused on the discovery of spontaneous reverse movement of tRNA, or reverse translocation in the ribosome. Despite the existing dogma that translation occurs in a single direction, tRNA and mRNA can spontaneously move in the reverse direction. Our results suggested that spontaneous translocation is reversible and endergonic depending on the codon context, supporting the earlier prediction that EF-G acts as a Brownian motor by inducing conformational changes of the complex that allow Brownian movement of tRNA to take place.This finding further helps elucidate the molecular mechanisms of antibiotic actions in collaboration with the Jamie Cate's group at University of California, Berkeley, as described in Chapter 3. Antibiotic spectinomycin traps the swiveling of the head domain of the 30S subunit and inhibits both forward and reverse translation, suggesting that the head swiveling is critical for the movement of tRNA in the ribosome. It is also found that hygromycin B induces distinct conformational changes in the 1492-1493 loop of the 30S A site among other aminoglycosides and inhibits reverse translocation. The results suggested that the flipping of the A1493 base in the distinct orientation sterically blocks the entry of P-site tRNA to the A site.In Chapter 4, I propose how the tRNA-ribosome interactions, particularly in the 30S P site and 50S E site, contribute to binding of tRNAs to the P site. The effects of several mutations in the 30S and 50S subunits on the dissociation (koff) and association rate ( kon) of various tRNA species in the P site are examined. Several 16S rRNA mutations affect binding of tRNAs to varying degrees and thus decrease the uniformity of tRNA binding, supporting the idea that tRNA-ribosome contacts in the 30S subunit are idiosyncratically tuned for uniform binding. Mutation C2394A of the 50S E site restores the uniformity in the presence of G1338U of the 30S P site, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. In addition, mutation C2394A or the presence of an N-acetyl-aminoacyl group decreases kon dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.Finally, Chapter 5 describes in vivo analysis of the lepA deletion mutant. LepA is an elongation factor that can catalyze reverse translocation in vitro. But the physiological role of LepA in bacteria is unclear. Here, deletion of the lepA gene (DeltalepA) in E. coli causes hypersensitivity to potassium tellurite and penicillin G, but has little or no effect on cell growth in the presence of superoxide radical generators paraquat and phenazine methosulfate. DeltalepA does not increase miscoding or frameshifting errors in the absence or presence of tellurite, indicating that LepA does not normally contribute to the fidelity of translation. These results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation.