Schilbach, S. | bioRxiv Science

bioRxiv2022

Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II

RNA Polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes such as small nuclear RNA (snRNA) genes is not understood at the structural level. Here we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol II preinitiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE) located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc called wing-1 and wing-2 bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

molecular biology↗

bioRxiv2021

Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 [A]. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

molecular biology↗