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Inhibition of protein synthesis is one of the most common modes of action for medically useful antibiotics. This thesis presents the mechanistic studies of two chemically distinct classes of antibiotics that target bacterial ribosomes –aminoglycosides and thermorubin. Arbekacin (ABK) and amikacin (AMK) are two new-generation semisynthetic aminoglycosides (AGAs) that were developed to overcome enzyme-mediated AGA resistance in bacteria. Our results demonstrate that these two antibiotics induce potent inhibitory effects on various phases of bacterial protein synthesis. The binding of ABK stalls elongating ribosomes to a state that is unfavorable for elongation factor-G (EF-G) binding, which drastically prolongs the time for translocation from ~50 milliseconds to at least 2 seconds. ABK also abolishes the accuracy of mRNA decoding and inhibits peptide release. The results of in vitro fast kinetics and structures of ABK and AMK-bound 70S ribosomes reveal that in addition to canonical binding at h44 of 16S rRNA, appended amino-hydroxy butyryl (AHB) moiety of ABK and AMK secures extra interactions at the binding pocket and provides long dwelling-time on the translating ribosome. Moreover, AMK binds at the large subunit of ribosome proximal to the 3'CCA-end of the tRNA in the P-site and inhibits the release factor-mediated peptide release. Our data suggest that AGA impose bacteriostatic effects mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors. We have further characterized the molecular mechanism of action of the antibiotic thermorubin (THB) using in vitro fast kinetics and cryo-EM. We found that THB impedes elongation, termination, and ribosome recycling phases of translation. THB does so by binding to the intersubunit bridge B2a and extruding C1914 of H69 of 23S rRNA that interferes with the interactions of A-site substrates including aminoacyl-tRNAs, class-I release factors, and ribosome recycling factor. We also found that THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. These studies altogether suggest that in-depth characterization of antibiotic action provides important clues that hopefully aid in the development of new antibiotics to fight against looming antibiotic resistance.

The ribosome is a large, ancient multicomponent macromolecular complex which is highly amenable to study by cryogenic electron microscopy (cryo-EM) and computation biology methods. This thesis delves into the structure of both prokaryotic and eukaryotic ribosomes in the context of determining a solution to emerging antimicrobial resistance. We show that thermorubin (THB) binds to the E. coli ribosome at intersubunit bridge B2a, flipping out 23S rRNA residue C1914 which interferes with A-site substrates. The position and rearrangements caused by THB also accounts for the biochemical results showing a decrease in elongation, termination and recycling phases of translation. Also using cryo-EM we looked at the Giardia intestinalis ribosome, determining six high-resolution structures representing translocation intermediates. Giardia is a protozoan parasite causing diarrhoea in humans, with metronidazole strains emerging. As the ribosome is often a target for antimicrobial drugs, work on the structure and function of the ribosome is of utmost important in determining an alternative therapeutic approach to the treatment of giardiasis. We also show naturally bound tRNAs and eEF2 on the Giardia ribosome, exhibiting eukaryote-specific subunit rolling and eEF2 with GDP in a uniquely positioned P_i primed for release, adding to the mechanism of translocation in protists as well as illustrating the evolution of both the structure and function of translation machinery. Finally, the molecular basis of thermostability in translational GTPases is explored using molecular dynamics of mesophilic and thermophilic elongation factor EF-Tu. Through ancestral sequence reconstruction two key interactions: in the GTPase domain; and an interdomain interaction were shown to be important in the overall structural stability of EF-Tu in high temperature environments. These studies together highlight the strength of utilising both structural and computational techniques to explore the translation apparatus.