A tunable CTD grammar governs the spatial programming of the transcription cycle

RNA polymerase II transcribes all protein-coding genes in eukaryotes, with its C-terminal domain (CTD) acting as a regulatory platform throughout the transcription cycle. Despite its simple sequence, the conserved heptad repeats encode a regulatory grammar critical for transcription initiation, elongation, and termination. Using structural analysis, single-molecule imaging, and synthetic CTD-engineered Pol II constructs, we define the minimal sequence requirements across transcriptional stages. We show that while SP motifs are essential phosphorylation sites, flanking residues can tolerate variability. In contrast, the periodic positioning of tyrosine residues is indispensable for pre-initiation complex (PIC) formation through interactions with the Mediator, which are disrupted upon Ser5 phosphorylation, triggering promoter escape. This supports a model where sequential tyrosine engagement stabilizes PIC assembly, and progressive phosphorylation propels Pol II escape. Site-specific Ser2/5 phosphorylation orchestrates 3'-end processing factor recruitment. These findings define the functional grammar of the Pol II CTD and explain how a low-complexity sequence achieves regulatory specificity.