No abstract

The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The mechanism of how the kinases place phosphorylation on the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinasesCDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.

No abstract

The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...