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

The carboxy-terminal domain (CTD) of RNA Polymerase II, composed of tandem heptad repeats with the consensus sequence YSPTSPS, orchestrates the transcription cycle through a dynamic series of post-translational modifications. Among these, the phosphorylation of Ser5 is critical for initiator/promoter clearance and the recruitment of capping enzymes. However, the exact conformational consequences of these modifications are still not fully understood. This study investigates how Ser5 phosphorylation affects the local and global conformation of the CTD, its influence on proline isomerization, and how variations in the repeat sequence modulate these effects. We employed Gaussian accelerated Molecular Dynamics (GaMD) simulations on 3-heptad models of both the consensus CTD sequence and an Asn7 variant. 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, which leads to compaction. We used a clustering algorithm to identify commonly populated conformations, with a focus on those conformations that change in population with Ser5 phosphorylation. Our simulations reveal that the expansion of the CTD due to Ser5 phosphorylation is accompanied by a change in local, intra-heptad interactions in both variants. Notably, phosphorylation significantly increases the population of cis-Pro6 due to steric repulsion between the Asn7 side chain and the large side chain of the phosSer5, but has a smaller increase in the consensus variant. These results clarify the underlying mechanisms by which phosphorylation can modulate the CTDs structural landscape to regulate the transcription cycle. SignificanceThe RNA Polymerase II CTD is a critical part of the machinery that regulates transcription, and therefore, understanding how it functions in this process is essential. However, the conformational effects of known modifications to the CTD, such phosphorylation and proline isomerization, are not fully understood. This paper uses all-atom molecular dynamics simulations to identify the specific conformational changes to the disordered CTD with phosphorylation, and with changing heptad sequence. We also identify the interactions that are responsible for these changes. Our results emphasize that two chemical properties of phosphate groups, their negative charge and their large size, can affect protein conformation. For the CTD, these properties have competing effects on the overall compaction of the disordered sequence.