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.Author summary: Gene expression in human cells is a carefully coordinated process that ensures the right genes are activated at the right time. Central to this process is RNA polymerase II, a multi-protein complex that transcribes DNA into precursors of messenger RNA. Its largest subunit – RPB1 – possesses a flexible tail, called the C-terminal domain (CTD), which acts as a platform for other proteins that help control transcription. To function properly, the CTD must be decorated with chemical marks, most importantly phosphate groups, by enzymes known as kinases. In our study, we focus on one of these kinases, called P-TEFb, which plays a key role during transcriptional elongation. Previous work suggested that P-TEFb adds phosphate groups in a random, distributive fashion, but it was unclear whether its activity depends on the surrounding phosphorylation state. Using mathematical models trained on experimental data, we find that P-TEFb tends to add new phosphate marks next to sites that are already modified. This means that its activity is locally cooperative rather than purely random. Our results suggest that the local chemical environment of the CTD helps guide the formation of phosphorylation patterns during gene transcription.