Quantitative modelling of P-TEFb mediated CTD phosphorylation identifies local cooperativity.

Fine-tuned regulation of RNA polymerase II (Pol II) activity is essential for accurate gene expression. A key layer of this regulation involves phosphorylation of Pol II's C-terminal domain (CTD), a repetitive heptapeptide tail that coordinates transcription and RNA-processing factors. The kinase P-TEFb plays a major role in this process, yet its precise phosphorylation mechanism remains unclear. Previous in vitro studies have suggested a distributive mode of action based largely on qualitative inspection of mass spectrometry data rather than quantitative analysis. Here, we use mathematical modelling of CTD phosphorylation to explore whether local context, such as neighbouring phosphorylations or directional biases, affects P-TEFb activity on the CTD. Our results indicate that P-TEFb acts distributively but with pronounced local cooperativity: repeats adjacent to phosphorylated sites are modified at higher rates. We find no evidence for directional bias, although the limited positional resolution of the data precludes a definitive conclusion. These results identify local context as an important factor in P-TEFb-mediated CTD phosphorylation and establish a quantitative modelling framework for dissecting multi-site modification dynamics.