Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Abstract

Quantifying the effect of vital resources on transcription and translation helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic transcription-translation (TX-TL) system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg²⁺), on gene expression. Recent observations of the counterintuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by transcription, hence leaving fewer resources for translation. In this work, we investigate an alternative hypothesis: direct suppression of the translation rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource limitation argument. To further decouple the contributions of transcription and translation, we performed gene expression experiments with purified mRNA. Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring translation rates for different Mg²⁺ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg²⁺ can partially reverse this inhibition. In several experiments, we observe two maxima of the translation rate viewed as a function of both Mg²⁺ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2⁺ titration effect. The non-trivial compensatory effects of abundance of different vital resources signals the presence of complex regulatory mechanisms to achieve optimal gene expression.