Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

Abstract

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. Here we identify a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalize on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms.

13C Direct Detected NMR for Challenging Systems

“…However, several kinases change their activity depending on pH and temperature and it is often important to investigate their effect in conditions near physiological ones. Therefore, the possibility to follow these reactions with an additional experiment, the CON, in particular if acquired simultaneously to the 2D HN, offers an excellent tool of investigation for this challenging topic. , Another strategy that has been proposed, in particular in cases in which sensitivity is an issue (either for low protein concentration or for instrumental setup) is the use of the most sensitive 2D experiment, the 1 H-start CACO. Several variants focusing on glycine or nonglycine residues were indeed proposed and used to follow phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4 …”