BPS2026 – RNA polymerase II CTD Ser5 phosphorylation induces competing effects of expansion and compaction

The carboxy-terminal domain (CTD) of RNA polymerase II is involved in modulating the transcription cycle over a dynamic series of post-translational modifications. This CTD consists of a repeating consensus heptad sequence (YSPTSPS) with some variable heptads. The phosphorylation of Ser5 is one such modification that enables the alternating expansion/compaction of the CTD, but this mechanism is not well understood. We have investigated the consequences of Ser5 phosphorylation on the local and global conformation of the CTD and its influence on proline isomerization within the structure, comparing two variations of the CTD sequence. A total of 40 Gaussian-accelerated molecular dynamics (GaMD) simulations were performed with 3-heptad models of the CTD sequence: one model with the consensus sequence in all three heptads, and one with an Asn-variant (YSPTSPN) in the middle heptad, and consensus on either side. We found that Ser5 phosphorylation promotes expansion of the peptide due to the repulsion between the negatively charged phosphate groups, but also increases the population of cis -Pro6, leading to compaction. Our simulations reveal that the expansion of the CTD associated with Ser5 phosphorylation is accompanied by a change in local, intra-heptad interactions in both variants. Phosphorylation significantly increases the population of cis -Pro6 due to steric repulsion between the Ser/Asn7 side chain and the large side chain of the phosSer5 in both variants. These results clarify the underlying mechanisms by which phosphorylation can modulate the CTD’s structural landscape to regulate the transcription cycle.