Fidelity of protein synthesis using sequence reconstructed ancient Elongation Factor Tu

Sammanfattning: To maintain a healthy growth rate of living cells, protein synthesis must take place accurately and efficiently. Translation, the core step decoding the genetic information, governs both fidelity and efficiency. For keeping a low error level, translation mainly relies on the codon-anticodon basepairing of mRNA and tRNA, which is known as ‘initial selection’. Apart from that, nature also evolved proofreading to reduce errors in protein synthesis. The current study focuses on initial selection of the correct tRNA.  EF-Tu, Elongation Factor Thermo-unstable, is an essential housekeeping GTPase factor responsible for transferring aa-tRNA to the ribosome. EF-Tu contributes to accuracy of initial selection although the exact mechanism is unknown. Here we have characterized and compared two sequence reconstructed ancestral EF-Tus, which are 1.3 and 3.3 billion years old respectively. Using dipeptide formation assay, we obtained the Michaelis-Menten parameters for Leu-tRNALeu on a near-cognate codon. Comparing the specificity parameter kcat/KM for the near-cognate vs. cognate we determine the accuracy of tRNA selection. My results show lower efficiency but higher accuracy using ancestral EF-Tus supporting the theory of trade-off between efficiency and accuracy. We envisage that during evolution EF-Tu sacrifices some accuracy to achieve higher efficiency as seen with modern EF-Tus.  My results demonstrate that EF-Tu can coordinate both the fidelity and the efficiency of translation.