Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components

Abstract

Thermus thermophilus is a thermophilic model organism distantly related to the mesophilic model organism E. coli . We reconstituted protein translation of Thermus thermophilus in vitro from purified ribosomes, transfer ribonucleic acids (tRNAs) and 33 recombinant proteins. This reconstituted system was fully functional, capable of translating natural messenger RNA (mRNA) into active full-length proteins at temperatures up to 65°C and with yields up to 60 μg/ml. Surprisingly, the synthesis of active proteins also occurred at 37°C, a temperature well below the minimal growth temperature for T. thermophilus . A polyamine was required, with tetraamine being most effective, for translation at both high and low temperatures. Using such a defined in vitro system, we demonstrated a minimal set of components that are sufficient for synthesizing active proteins at high temperatures, the functional compatibility of key translation components between T. thermophilus and E. coli , and the functional conservation of a number of resurrected ancient elongation factors. This work sets the stage for future experiments that apply abundant structural information to biochemical characterization of protein translation and folding in T. thermophilus . Because it contains significantly reduced nucleases and proteases, this reconstituted thermostable cell-free protein synthesis system may enable in vitro engineering of proteins with improved thermostability.

Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein

“…1). Furthermore, the ancient EF-Tu protein exhibits the closest phenotypic property to the endogenous EF-Tu in terms of observed melting temperature (Tm) and its activity in a reconstructed in vitro translation machinery in which all other components necessary for translation besides EF-Tu are provided from the contemporary translation machinery (Gaucher et al 2008; Zhou et al 2012). This suggests that co-evolution between EF-Tu and aa-tRNAs/ribosome/nucleotide-exchange-factors in E. coli since the divergence of the ancestral and modern EF-Tu forms has not prevented the ancestral EF-Tu from interacting with the modern E. coli translation components (Kacar and Gaucher 2012).…”

Experimental evolution of Escherichia coli harboring an ancient translation protein

“…1). Furthermore, the ancient EF-Tu protein exhibits the closest phenotypic property to the endogenous EF-Tu in terms of observed melting temperature (Tm) and its activity in a reconstructed in vitro translation machinery in which all other components necessary for translation besides EF-Tu are provided from the contemporary translation machinery (Gaucher et al 2008;Zhou et al 2012). This suggests that co-evolution between EF-Tu and aa-tRNAs/ribosome/nucleotide-exchange-factors in E. coli since the divergence of the ancestral and modern EF-Tu forms has not prevented the ancestral EF-Tu from interacting with the modern E. coli translation components (Kacar and Gaucher 2012).…”

A Strategy for Designing Thermostable Enzymes by Reconstructing Ancestral Sequences Possessed by Ancient Life

“…Then, they observed that the thermophilic ancestor adapted to cooler environment progressively as the temperature of the ancient ocean decreased (Gaucher et al 2008 ). In addition, the reconstructed elongation factor Tu works correctly in an extant thermophilic organisms (Zhou et al 2012 ). Similarly, Hart et al ( 2014 ) investigated how modern mesophilic and thermophilic ribonuclease H1 (RNH) have adapted to low and high temperatures, respectively.…”