show Abstracthide AbstractThe Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase (Pol) II. Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here, we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. Consistent with a model of condensate-driven transcription initiation, large clusters of hypo-phosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected, CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates genome-wide. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell type-specifying transcriptional networks. Overall design: PRO-seq in KBM7 MED14-dTAG cells after 1-2h 500nM dTAG7 and/or 30min 500nM NVP2.