Phosphorylation modulates secondary structure of intrinsically disorder regions in RNA polymerase II.

The intrinsically disordered C-terminal domain (CTD) of RNA polymerase II contains tandem repeats with the consensus sequence YSPTSPS and coordinates transcription and co-transcriptional events through dynamic phosphorylation patterns. While it has been long hypothesized that phosphorylation induces structural changes in the CTD, a direct comparison of how different phosphorylation patterns modulate the CTD conformation has been limited. Here, we generated two distinct phosphorylation patterns in an essential Drosophila CTD region with the kinase Dyrk1a: one where Ser2 residues are primarily phosphorylated, mimicking the state near transcription termination, and a hyperphosphorylation state where most Ser2, Ser5, and Thr residues are phosphorylated, expanding on our work on Ser5 phosphorylation, which mimics early transcription elongation. Using 13C Direct-Detect NMR, we show that the CTD tends to form transient beta strands and beta turns, which are altered differently by Ser2 and Ser5 phosphorylation. Small angle x-ray scattering (SAXS) revealed no significant changes in the CTD global dimensions even at high phosphorylation levels, contradicting the common assumption of phosphorylation-induced chain expansion. Our findings support a transient beta model in which unphosphorylated CTD adopts transient beta strands at Ser2 during transcription pre-initiation. These transient structures are disrupted by Ser5 phosphorylation in early elongation, and later restored by Ser2 phosphorylation near termination for recruiting beta turn-recognizing termination factors.