Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets

Efficient gene expression requires RNA Polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in 3D nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. Here, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation, and reduces transcription bursting. This study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.