Structural and biochemical characterization of RNA polymerase II transcription

Abstract

Eukaryotic development requires precise temporal regulation of gene expression orchestrated through a series of complex mechanisms. One such mechanism involves pausing of RNA polymerase II (Pol II) in the promoter-proximal region of genes. Pausing is stabilized by the protein complexes DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF). Prior structural and biochemical studies provide specific mechanisms for stabilization of paused Pol II by NELF. However, cellular data suggests that NELF can accompany actively elongating Pol II into the gene body, indicating that NELF may be able to associate with pol II without enforcing Pol II pausing. This thesis presents cryo-electron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations on the surface of Pol II, the paused state and the poised state. The poised state does not support a tilted RNA-DNA hybrid, a key characteristic of pausing, indicating that NELF in the poised state is compatible with elongating Pol II. Furthermore, Pol II bound to NELF in the poised conformation can accommodate TFIIS binding simultaneously, allowing reactivation of Pol II at sites of pausing or backtracking. Finally, a region of the flexible NELF-A tentacle engages with the RPB2 protrusion, an interface necessary for pausing. These results define how NELF can support both Pol II pausing and elongation and provide the molecular basis for how transcription can be reactivated when NELF is bound to Pol II.