Pyrrolysine supply and demand shapes the translational landscape in Methanosarcina acetivorans

The amber stop codon (UAG) can encode for pyrrolysine (Pyl) or be read as a stop codon by methylamine-metabolizing organisms including methanogenic archaea. The fate of UAG is decided during the decoding step of translation by competition between the pyrrolysine-aminoacylated transfer RNA (Pyl-tRNA Pyl ) and release factor. To further understand the consequences of pyrrolysine based genetic code expansion, we integrated RNA sequencing, tRNA charging analysis, and codon-resolved mono- and disome ribosome profiling in Methanosarcina acetivorans . During conditions of high pyrrolysine demand, we observed increased expression of the pyrrolysine biosynthetic operon with a concurrent increase in acylation of tRNA Pyl and ribosome occupancy at UAG codons. During low pyrrolysine demand, the population of Pyl-tRNA Pyl decreases and we observe a strong ribosome pausing signal during UAG decoding. We find that the dwell time on UAG codons is shorter during high demand, but ribosome collisions increase due to greater ribosome density on UAG-containing transcripts. Together, these results show how pyrrolysine demand modulates tRNA charging and controls elongation dynamics, clarifying the cellular consequences of decoding an ambiguous stop codon.