Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from “Generalist” Ancestors

Abstract

It has been hypothesized that early enzymes are more promiscuous than their extant orthologues. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using Elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing pre-steady state fast kinetics using quench-flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3 to 3.3-billion-year-old ancestral EF-Tus towards two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. While the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. Also, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remain virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, ‘generalist’ ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.

Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins

“…Other distantly related nitrogenase-like homologs have putative catalytic roles, including a recently reported homolog suggested to participate in a methionine salvage pathway that forms ethylene (North et al 2020). It is possible that a maturase-like predecessor would have been promiscuous, a suggested general feature of early-evolved proteins (Copley 2015; De Tarafder et al 2021), and capable of both providing a scaffold for cluster maturation as well as catalysis using the same matured cluster. Gene duplication and divergence might then have subsequently specialized maturases to only function as a scaffold and evolve residues that permit the release of the matured cluster.…”

Early divergence of translation initiation and elongation factors

“…It is generally accepted that the ribosome and its protein factors were already established in LUCA 4,6 . Expansion of the translation machinery, on the other hand, is thought to have occurred prior to LUCA 68 potentially following the rRNA core center of the ribosome formation, 31,69 strengthening the hypothesis that, unlike the ribosome, 70 the supporting translational factors were not yet fully established in the early cellular era. While we do not know if LUCA had adapted a system that is largely similar to the extant translation machinery, or whether translation‐as‐we‐know‐it subsequently emerged in a stepwise fashion or through simultaneous maturation of supporting components, 71,72 tracing the evolutionary history of early translation factors will unravel the fascinating story of life's core information processing machinery.…”

“…The other copy, the EF‐Tu ancestor, coevolved with tRNAs, gaining flexible conformational function in the GTP‐binding domain, and acquired mutations to bind a wider variety of tRNA molecules, specializing for EF‐Tu functionality. Intriguingly, it was recently shown that ancestral EF‐Tu proteins were generalists in terms of their compatibility with ribosomes, compared to the modern EF‐Tus 31 . The translation system consists of numerous essential molecules and the complete picture of evolutionary trajectory of translation over geologic time will require an integrated view into other components such as the other translation GTPases (e.g., EF‐G, RF3) and tRNAs 67 with the underlying chemical systems.…”

“…Intriguingly, it was recently shown that ancestral EF‐Tu proteins were generalists in terms of their compatibility with ribosomes, compared to the modern EF‐Tus. 31 The translation system consists of numerous essential molecules and the complete picture of evolutionary trajectory of translation over geologic time will require an integrated view into other components such as the other translation GTPases (e.g., EF‐G, RF3) and tRNAs 67 with the underlying chemical systems. Further, ancestral tRNA‐IF2 interactions may differ from those of extant translation interaction partners.…”

“… 30 The shared key features include GTPase activity and tRNA binding in both IF2 and EF‐Tu proteins, leading to a parsimonious explanation that these two proteins descended from an ancient, common ancestral protein. Other studies have shed light on the ancestry of EF‐Tu 31 , 32 , 33 , 34 as well as its diverse role in extant cells. 16 Intriguingly, EF‐Tu was suggested to be the first GTPase in early translation machinery.…”