Structure of paused transcription complex Pol II-DSIF-NELF

Metazoan gene regulation often involves pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy (cryo-EM) structure of the paused Sus scrofa/Homo sapiens Pol II-DSIF-NELF transcription elongation complex (PEC) at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate (NTP) substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing factor TFIIS. Additionally, NELF possesses two flexible tentacles that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding NELF function during promoter-proximal gene regulation.