Aminoacyl-tRNA synthetases (aaRSs) serve a dual role in charging tRNAs. Their enzymatic activities both provide protein synthesis flux and reduce uncharged tRNA levels. Although uncharged tRNAs can negatively impact bacterial growth, substantial concentrations of tRNAs remain deacylated even under nutrient-rich conditions. Here, we show that tRNA charging in Bacillus subtilis is not maximized due to optimization of aaRS production during rapid growth, which prioritizes demands in protein synthesis over charging levels. The presence of uncharged tRNAs is alleviated by precisely tuned translation kinetics and the stringent response, both insensitive to aaRS overproduction but sharply responsive to underproduction, allowing for just enough aaRS production atop a “fitness cliff.” Notably, we find that the stringent response mitigates fitness defects at all aaRS underproduction levels even without external starvation. Thus, adherence to minimal, flux-satisfying protein production drives limited tRNA charging and provides a basis for the sensitivity and setpoints of an integrated growth-control network.

氨酰 tRNA 合成酶 (aaRS) 在为 tRNA 充电中起双重作用。它们的酶活性既提供蛋白质合成通量，又降低不带电荷的 tRNA 水平。尽管不带电的 tRNA 会对细菌生长产生负面影响，但即使在营养丰富的条件下，大量 tRNA 仍会脱酰。在这里，我们展示了枯草芽孢杆菌中的tRNA 充电由于在快速生长期间优化了 aaRS 的产生，因此没有最大化，这将蛋白质合成的需求置于充电水平之上。不带电荷的 tRNA 的存在通过精确调整的翻译动力学和严格的反应得到缓解，两者都对 aaRS 生产过剩不敏感，但对生产不足反应迅速，允许在“健身悬崖”上产生足够的 aaRS。值得注意的是，我们发现即使没有外部饥饿，严格的响应也可以减轻所有 aaRS 生产不足水平的适应度缺陷。因此，坚持最小的、满足通量的蛋白质生产会驱动有限的 tRNA 充电，并为集成生长控制网络的灵敏度和设定点提供基础。