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.
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.
New England Biolabs, Inc, 240 County Road, Ipswich, MA 01938, USA.
Ying
Y
Zhou
Haruichi
H
Asahara
Eric A
EA
Gaucher
Shaorong
S
Chong
D001426:Bacterial Proteins; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006358:Hot Temperature; D010445:Peptide Elongation Factors / Q000378:metabolism; D011073:Polyamines / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012269:Ribosomal Proteins / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D010445:Peptide Elongation Factors; D011073:Polyamines; D012269:Ribosomal Proteins