Regulation of Promoter Proximal Pausing of RNA Polymerase II in Metazoans

Abstract

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

Keywords: DSIF; NELF; P-TEFb; Pol II; TFIID.

Figure 1:. Surface representation of paused Pol II-DSIF-NELF complex structure.

A) Side view of complex illustrating modeled path of the unstructured NELF-A C-terminal tentacle (green)[72]. B) Top view of complex illustrating modeled path of the unstructured NELF-E C-terminal tentacle (blue)[72]. PDB ID: 6GML.

Figure 2:. Spt5 DNA and RNA clamps.

A) Superimposed structures of the Spt5 nucleic acid clamps in their paused (grey, PDB ID: 6GML) and active (orange, PDB ID: 6GMH). The KOW1 and KOW4 domains change conformation upon pause release, resulting in clamp opening. B) Surface model of Pol II-DSIF. The Spt5 NGN and KOW1 domains (shown in orange) form a clamp around the upstream DNA (purple) and the Spt5 KOW4 and KOW5 domains (orange) form a clamp around the nascent RNA (blue). PDB ID: 6GML.

Figure 3:. Half-translocated RNA-DNA duplex in the paused RNA polymerase active site.

A) The RNA-DNA duplex observed in the Pol II-DSIF-NELF structure (colored, PDB ID: 6GML) is in a “tilted” conformation relative to the RNA-DNA duplex observed in the Pol II-DSIF structure (grey, PDB ID: 5OIK). B) The RNA-DNA hybrid in the active site of the paused bacterial RNA polymerase (colored, PDB ID: 6ASX) adopts a similarly tilted conformation compared to the hybrid in the active site of a post-translocated RNA polymerase (grey, PDB ID: 6ALF).

Figure 4:. Conserved Spt5 and NELF P-TEFb phosphorylation sites.

Ten of the residues identified as putative P-TEFb phosphorylation sites on human Spt5 are conserved or semi-conserved (Ser or Thr) in Drosophila Spt5. In contrast, only one putative NELF-A phosphorylation site is semi-conserved from human to Drosophila and three putative NELF-E phosphorylation sites are conserved from human to Drosophila. The S666 and T806 in Spt5 (red) have been identified as critical participants in the phosphatase-kinase switch that governs pausing and pause release dynamics[78]; both residues are conserved in Drosophila.

Figure 5:. Summary of the factors involved in promoter proximal pausing.

A) Pol II pausing occurs ~30–60 nt downstream of the transcription start site and requires DSIF and NELF, which function cooperatively; TFIID helps promote pausing[59]. The pre-EJC helps maintain pausing at genes with long introns[62]. Additional “Factor X” proteins may contribute to pause stabilization. B) TFIID, perhaps in a different conformation than in (A), helps recruit P-TEFb and the SEC to the elongation complex[104]. H3K9ac can also facilitate recruitment of the SEC[140], a process that can be inhibited by histone deacetylases[139, 141]. C) The phosphatase PP4 promotes pausing by maintaining the dephosphorylated state of DSIF[78]. P-TEFb inactivates PP4 through phosphorylation and phosphorylates DSIF, NELF, and potentially phosphorylates the Pol II CTD. This results in the ejection of NELF from the elongation complex. For some genes, pause release can be triggered by enhancer activation, which is in turn inhibited by PAF1C[126]. D) Pause release is accompanied by association of elongation factors like Spt6 and PAF1C with the elongation complex, allowing the transition into productive elongation[73, 74]. TFIIS binds and rescues backtracked Pol II (not shown)[128, 129]. E) At human and some Drosophila promoters, the +1 nucleosome acts to stabilize paused Pol II[30, 31]. In Drosophila, FACT promotes pausing, possibly by stabilizing the +1 nucleosome[127]. In humans, SNF2H promotes nucleosome-stabilized pausing[30]. In the absence of pausing factors, nucleosomes act as barriers to productive transcription. These barriers can be relieved by chromatin remodelers SWI/SNF[135].