“Zipper” Grammar of CTD Governs the Spatial Programing of the Transcription Cycle of RNA Polymerase II [ChIP-Seq]

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 defined the minimal sequence requirements across transcriptional stages.

We show that while SP motifs are essential phosphorylation sites, flanking residues tolerate variability. In contrast, the periodic positioning of tyrosine residues is indispensable for pre-initiation complex (PIC) assembly through interactions with the Mediator, which are disrupted upon Ser5 phosphorylation triggering promoter escape. This supports a “zipper” model where sequential tyrosine engagement stabilizes PIC assembly and progressive phosphorylation propels Pol II escape.