The Complex Evolutionary History of EF-Tu

Abstract

Translation is a conserved and highly dynamic process that relates all domains of life. This informational processing system is mediated by a core network of translation machinery, which is hypothesized to have been present in the last universal common ancestor of bacteria, archaea, and eukarya and has tightly co-evolved throughout time. Specifically, elongation factor thermal unstable, or EF-Tu, is a universally conserved translational GTPase (trGTPase), highly expressed and essential in translation. The function and efficiency of translation elongation is critical, and therefore the dysregulation has been associated with health and disease in humans, including tumorigenesis and neurodegeneration. In this thesis, I analyze the evolution of EF-Tu, specifically focusing on ways EF-Tu may compensate and adapt in times of cell stress. In Chapter 2, we constructed the phylogeny and shared evolutionary history of essential trGTPases EF-Tu (aEF-1A/eEF-1A) and initiation factor IF2 (aIF5B/eIF5B). Our results show that the GTP-binding domain and tRNA-binding domain of the reconstructed common ancestor (IF2/EF-Tu) is more similar in protein sequence to the modern E. coli IF2, while the domain organization and structure of IF2/EF-Tu’s binding pocket is more comparable to modern EF-Tu. This work indicates that analogous to modern IF2, IF2/EF-Tu may have exhibited limited conformational change between GTP bound/unbound states. Whether the shared IF2/EF-Tu common ancestor was able to conduct both initiation and elongation functions requires further investigation. In Chapter 3, we experimentally evolved an engineered E. coli strain 3000 generations, in which modern EF-Tu was substituted for ancestral EF-Tu (AnEF) and discovered a synonymous mutation in the 5’ region of AnEF (C45T). This synonymous mutation, which appeared after 2,000 generations, is correlated with an increase in AnEF protein and mRNA levels, as well as an increase in polysome abundance during translation. However, the observed increase in population growth fitness was seemingly contingent on the presence of one or more additional mutations in the evolved genetic background. In Chapter 4, we discovered a tuf-like gene, which encodes for EF-Tu, in 3 megaplasmids which are found in and have been independently acquired by various unrelated Pseudomonas strains spanning multiple species. To study these megaplasmid tuf genes, we constructed EF-Tu phylogenies and found that the megaplasmid EF-Tu has a single point of origin mapped within the clade of Pseudomonas. Furthermore, we compared megaplasmid EF-Tu peptide sequence and predicted structures to their chromosomal counterparts and observed a divergence in the protein sequence, yet a high degree of conservation in protein structure relative to P. syringae EF-Tu. This work reports the first time a tuf gene was identified in a bacterial megaplasmid.