Hierarchical buffering of cellular RNA polymerase II pools maintains transcriptional homeostasis

RNA polymerase II (Pol II) abundance and transcriptional output are coordinated across diverse conditions to maintain mRNA homeostasis. To determine whether Pol II acts as a limiting factor for transcription, we combined acute perturbations with quantitative single-cell imaging and genome-wide profiling. We find that transcription remains stable across 70-180% of normal Pol II levels in human cells. This robustness emerges from hierarchical buffering mechanisms operating on distinct timescales. Within tens of minutes after monoallelic depletion of Pol II subunit POLR2A, cells maintain steady transcriptional output by drawing polymerases from a nucleoplasmic reserve pool. However, over hours, cellular Pol II holoenzyme levels also recover, driven by constitutive excess POLR2A subunit production rather than feedback sensing. Finally, strong depletion over days activates additional compensatory mechanisms. Together, these results reveal how temporal separation of buffering mechanisms provides robustness to perturbations of essential machinery, illuminating how cells maintain homeostatic control of essential complexes.