Elucidating the Bell-Shaped Dependence of Protein Translation Activity on EF-Tu Concentration in a Reconstituted Cell-Free System Using a Mechanistic Model

Protein synthesis in cell-free protein synthesis systems often exhibits non-intuitive input-output relationships. In the PURE system, a reconstituted cell-free system, protein production peaked at low elongation factor Tu (EF-Tu) concentrations and decreased at higher concentrations, resulting in a characteristic bell-shaped profile. Here, we investigated the origin of this behavior using a detailed mechanistic model of translation in the PURE system, designated as ePURE, which describes reaction dynamics of hundreds of molecular species and reactions. Our computational analysis suggested that excess EF-Tu sequesters the initiator tRNA (tRNA ^fMet ) into non-productive EF-Tu·GTP·Met-tRNA ^fMet complexes, thereby depleting the pool of initiator tRNA available for translation initiation. This suppression arises from competition for a limited molecular resource rather than from direct inhibition. Based on this mechanism, we predicted that increasing the concentrations of tRNA ^fMet and methionyl-tRNA formyl-transferase would eliminate the bell-shaped dependence, and experimentally confirmed this prediction. Under these modified conditions, the bell-shaped response disappeared and protein production was enhanced. These findings demonstrate how mechanistic computational models can reveal hidden constraints underlying non-intuitive input-output relationships in complex biochemical networks and guide the rational optimization of cell-free protein synthesis systems.