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.
Highlights
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
When a transgenic fly line expressing RNA-mediated interference (RNAi) against endogenous Rpb[1] in response to the GAL4 activator is mated to a fly line that ubiquitously expresses GAL4, no adult progeny are produced. This lethality is overcome by co-expressing a wild-type version of Rpb[1] that has been rendered resistant to the RNAi through synonymous substitutions in the Rpb1-coding sequence (Fig. 1a)
To identify regions of the CTD essential for the development of an adult fly, several deletions were made in the CTD of the RNAi-resistant form of Rpb[1] and tested for their ability to rescue the lethality associated with depleting the endogenous Rpb[1] (Fig. 1b, Supplementary Fig. 1b)
Summary
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. The carboxy-terminal domain (CTD) of Rpb[1], the largest subunit in RNA polymerase II, is an essential regulator of eukaryotic gene expression. This intrinsically disordered protein (IDP), consisting of multiple tandem repeats of the consensus sequence (Y1S2P3T4S5P6S7), acts as a scaffold for the recruitment of factors required for transcription, mRNA biogenesis and modification of the chromatin structure[1]. We turn to Drosophila, which is unique among commonly used model organisms in that only 2 of its 42 heptads precisely match the consensus sequence This feature has allowed us to use NMR and mass spectrometry to precisely monitor PTM patterns and local structural features in the context of a natural CTD sequence
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