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Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-Electron Microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal-proteins, but retain some bacterial features. The translocation intermediates, with naturally-bound tRNAs and eEF2, display characteristic ribosomal intersubunit rotation and small subunit’s head swiveling - universal for translocation. In addition, we observe the eukaryote-specific ‘subunit rolling’ dynamics, albeit with limited features. Finally, the eEF2•GDP state features a uniquely positioned ‘leaving Pi’ that proposes hitherto unknown molecular events of Pi- and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes both from the structural and mechanistic perspectives.

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

The molecular basis of protein thermostability is diverse and unclear. To better understand it, we solved high-resolution crystal structures of four 0.5 – 3.5 billion year old ancestral bacterial Elongation Factor-Tus (EF-Tu). Structural comparison revealed two key interactions, unique for the thermophilic EF-Tus; i) a hydrogen bond between a G-domain tyrosine and the α- phosphate of the guanine nucleotide that stabilizes GTP/GDP; and ii) an inter-domain salt-bridge, tethering the Domains II and III via an arginine and a glutamic acid, respectively. We could reverse the thermostability profiles of the thermophilic and mesophilic EF-Tus by adding or removing these interactions, which were validated with aggregation, biophysical, and functional assays. Further, molecular dynamics simulations demonstrated that these interactions contribute to thermostability via the Mg2+ in the nucleotide binding site. Furthermore, the inter-domain interaction restricts the transition between the open and the closed conformations of the EF-Tus, thereby regulating their thermoactivity.