The process of protein synthesis via translation is of paramount importance for the existence of life on Earth. The bacterial translation machinery has embraced more than 3.5 billion years of molecular evolution to adapt and function efficiently under the provided physiological conditions. This thesis dwells on the intricacies of the adaptive evolution, which the massively complex translation machinery has undergone to function optimally in diverse conditions and habitats. In Paper I, we used elongation factor Tu (EF-Tu) as a model system to follow the evolution of ribosome specificity in translation factors. For that, we have biochemically characterized two sequence-reconstructed ancestral EF-Tu variants for their specificities towards two unrelated extant bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Our fast kinetics-based biochemical analysis hints at the ‘generalist’ ancestry of modern EF-Tu proteins. In Paper II, we have reconstituted an in vitro translation system of the psychrotolerant bacteria Pseudoalteromonas haloplanktis to quantitatively characterize the steps of translation elongation. Our results demonstrate similar kinetics of peptide bond formation in psychrotolerant P. haloplanktis and mesophilic E. coli. In contrast, P. haloplanktis ribosome exhibits much slower rates of EF-G-catalyzed tRNA translocation than E. coli. Comparison and swapping of the EF-Gs and tRNAs between the two in vitro translation systems indicate that the slow translocation is likely an inherent property of the P. haloplanktis ribosome. Furthermore, our results demonstrate the varied extent of antibiotic inhibition on the P. haloplanktis minimal translation system, particularly when targeting processes related to translocation and peptide bond formation, compared to E. coli. In Paper III, we used ribosomes from bacterial species of diverse habitats to show that the ribosomes in vitro can maintain their catalytic activity beyond the survival temperature cutoff of the native host. Moreover, our results indicate that the thermostability of essential translation factors, EF-Tu and EF-G, dictates the upper limit of reaction temperature for translation elongation. Finally, we demonstrate that ribosomes from a psychrophile, mesophile, and thermophile can function in a vast temperature range of 10-70 °C, provided the translation factors remain structurally and functionally stable. Our results highlight the thermal versatility of the ribosome and reiterate the early emergence of a thermostable ribosomal core in the primordial RNA world.

The outcome of this thesis will unveil some of the intricate mechanisms underlying the evolution of bacterial translation machinery. This knowledge may open up new research avenues regarding the emergence and diversification of bacteria and the development of new therapeutic strategies.