UC Berkeley

Ribosomes are macromolecular machines that catalyze cellular protein synthesis. While ribosome structure is well understood in model organisms, we lack a holistic understanding of how ribosome structure and adaptation has changed across divergent organisms. This thesis investigates structural changes in ribosomes and ribosome-binding proteins across prokaryotic species. Chapter 1 reviews our current understanding of ribosome structure, function, and adaption and their relevance in ribosome engineering.Chapter 2 examines rare rRNA sequence variation in the A loop, the binding site for the A-site tRNA in the LSU, found in a subset of hyperthermophilic archaea. In organisms from the archaeal phylum Thermoproteota, two highly conserved uridines in the A loop are instead cytosines. Substitution of these two positions with cytosines in the E. coli ribosome globally increased the stability of the LSU compared to wild type (WT), without affecting the activity of E. coli ribosomes. This sequence variation provides a strategy to stabilize the ribosome for future ribosome engineering efforts.Chapter 3 identifies and addresses a folding defect in ribosomes caused by a common rRNA tagging strategy in the LSU. Insertion of an MS2 tag into 23S rRNA helix H98 disrupts a key tertiary RNA contact, destabilizing the LSU. Guided by this insight, we designed a modified MS2 tag that restores H98 tertiary contacts. An analysis of ribosome structures revealed that many bacterial species form tertiary contacts between 23S rRNA helices near the 5′ and 3′ ends, which likely aid the stability of ribosomes across species.Chapter 4 explores divergent peptidyl transferase center (PTC) sequences in archaeal ribosomes. We found that archaeal PTC sequences are less conserved than those of bacteria. We determined the cryo-EM structure ribosomes from an archaeon with a divergent PTC, Pyrobaculum calidifontis. This structure revealed how divergent PTC sequences are accommodated, enabled by differences in bacterial and archaeal rProteins. We also identified a previously undescribed ribosome hibernation factor bound to the P. calidifontis ribosome, which we named Dri. Dri comprises a family of archaeal-specific ribosome hibernation factors distributed across multiple archaeal phyla.Chapter 5 continues the investigation of archaeal ribosome hibernation factors. We investigated divergent Dri homologs from the methanogenic archaeon Methanosarcina acetivorans. In the M. acetivorans genome, there are four archaeal ribosome hibernation factors encoded by adjacent genes and named them SriA-D. Cryo-EM structures of the M. acetivorans ribosome showed that SriA and SriB bind to the LSU and SriC and SriD bind to the SSU to inactivate the ribosome. A phylogenetic analysis revealed that Sri proteins broadly co-occur across archaea phyla.Finally, Chapter 6 presents the conclusion and outlines future directions for ribosomal structural biology across the tree of life.