An IDR-dependent mechanism for nuclear receptor control of mediator interaction with RNA polymerase II

We investigated modulation of metazoan MED interaction with RNA polymerase II (Pol II) by antagonistic effects of the MED26 subunit and the Cdk8 kinase module (CKM), both of which associate dynamically with MED. Results from in vivo studies point to a general and important role of MED26 in transcription, connected to Pol II recruitment. Analysis of CKM-MED and MED26-MED complexes by cryo-EM and crosslinking-mass spectrometry showed that CKM and MED26 have opposite effects on mMED association with Pol II (blocking and facilitating it, respectively), and revealed that the structural basis for their antagonistic interaction with MED relates to extended intrinsically-disordered regions (IDRs) in MED26 and the CKM subunit MED13 competing for interaction with MED. We found CKM-MED to be the preferred target of nuclear receptors (NRs), whose binding can initiate rearrangements of the MED13 IDR that allow MED26-dependent interaction of CKM-MED with the Pol II carboxy-terminal domain (CTD). Our results suggest that activators might play a critical role independent of MED recruitment, instead “activating” CKM-MED for CTD interaction in a MED26-dependent manner and, thus, controlling the start of preinitiation complex assembly. The presence of an intermediate state in which MED26 and the CTD II can interact with CKM-MED suggests a mechanism for fast, recruitment-independent activation connected to promoter-proximal Pol II pausing, with an activator enabling interaction between the originally inactive CKM-MED and a paused Pol II poised to initiate transcription. We found that CTD interaction with CKM-MED influences the subunit composition of the Tail module and this allostery suggests that the mechanism we propose could apply to non-NR activators targeting Tail subunits. Overall design: spike-in control mRNAseq, chipseq, CUT&RUN