A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis, to couple metabolism to cell cycle progression.

Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that cells regulate nitrogen (amino acid) and carbon metabolic homeostasis through tRNA U34-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in metabolic a starvation-signature. Thiolation mutants have only minor translation defects. However, these cells exhibit strongly decreased expression of phosphate homeostasis genes, mimicking a phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, and balance amino acid and carbon metabolic flux to maintain metabolic homeostasis, by controlling phosphate availability. These results further biochemical explain how phosphate availability determines a switch to a 'starvation-state'. Overall design: Ribosome-footprinting and RNA-seq samples of yeast CEN.PK, ?uba4 and ?ncs2 strains.