Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

Abstract

Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene regulation involve the pausing of RNA polymerase II (Pol II) in early elongation, and the controlled release of paused polymerase into productive RNA synthesis. Here we describe the factors that enable pausing and the events that trigger Pol II release into the gene. We also discuss open questions in the field concerning the stability of paused Pol II, nucleosomes as obstacles to elongation, and potential roles of pausing in defining the precision and dynamics of gene expression.

Keywords: Pol II pausing; gene expression; gene regulation; transcription regulation.

Figure 1.

Outline of early steps in the transcription cycle. Promoter DNA is shown with the TSS depicted by an arrow. TF-binding motifs and core promoter sequences are designated, with the average position of the first nucleosome shown, ∼140 bp downstream from the TSS. Distinct protein factors and complexes are involved in each step as labeled in the figure. Phosphorylation of DSIF (dichloro-1-β-D-ribofuranosylbenzimidazole [DRB] sensitivity-inducing factor), NELF and the Pol II CTD is shown as a green “P.” Active positive transcription elongation factor b (P-TEFb) is typically found within the superelongation complex (SEC) as shown. Elongation factors that facilitate productive elongation through chromatin include Spt6, FACT, and polymerase-associated factor 1 (PAF1).

Figure 2.

Rate constants favor the establishment of paused Pol II. The steps leading toward productive elongation and the relative rates of transitions between each step are depicted, with thicker arrows indicating faster rates. Note that Pol II in the PIC and paused forms are not coincident on the same DNA due to steric hindrance. The accumulation of Pol II in early elongation at most promoters indicates that the relative rates toward establishing paused Pol II (recruitment, initiation, and promoter escape) are typically faster than rates of Pol II release from pausing into productive elongation or premature termination. Upon termination, Pol II releases nascent RNA, which is rapidly degraded, and the polymerase is recycled into the pool of free Pol II.

Figure 3.

Core promoter elements and sequence features that lead to efficient promoter-proximal pausing. (A) TFs bind their sites and, with the help of the core promoter elements, recruit the transcription machinery to the promoter. DNA elements enriched in core promoters that promote PIC assembly include the TATA-box (−28 to −35 with respect to the TSS), Initiator (−4 to +2), MTE (+18 to +29), and DPE (+28 to +32) motifs. Drosophila promoters enriched in paused Pol II display a variant of the DPE called the pause button (PB, +25 to +35). Many of these core promoter elements play a dual role in initiation and pausing. The region around the pause site (+20 to +50) often contains sequences that produce locally strong RNA/DNA hybrids (red–yellow box) that can induce pausing and polymerase backtracking. A high GC skew in promoters and enrichment of G bases in the nontemplate strand of pause sites, especially in mammals, can lead to efficient pausing by creating strong RNA/DNA hybrids or possibly DNA and RNA structures that favor pausing. (B) Depiction of how strong RNA/DNA hybrids and pausing factors lead to pausing and backtracking of Pol II. After Pol II transcribes past a sequence with a strong RNA/DNA hybrid (red-yellow line) relative to the downstream sequence, the polymerase can pause. During pausing, the polymerase often backtracks such that the stronger more stable RNA/DNA hybrid is held within the Pol II active site. This movement causes the 3′ end of the RNA to be extruded though the funnel (gray), preventing further transcription elongation. NELF (orange) occludes the binding site of TFIIS (green) on the outer surface of Pol II, preventing TFIIS from reaching into the funnel to stimulate cleavage of the backtracked RNA and realignment of the RNA 3′ end with the polymerase active site.

Figure 4.

Pausing can serve as a mechanism to integrate multiple signaling events. TFs can stimulate initiation, elongation, or both. (A) When a signal responsive activator only stimulates elongation, for example, by recruiting P-TEFb, activation does not occur since polymerases were not initiated at the promoter prior to the TF binding. (B) If a signaling pathway and TF stimulates initiation only, then polymerases will pause proximally. Low levels of pause escape, possibly stimulated by Mediator-dependent P-TEFb recruitment, establishes a basal level of transcription. (C) When both signals are active, high levels of initiation and pause escape lead to robust transcriptional activation. By altering the level of input from either signaling pathway, the cell can fine tune the transcription levels of genes.

Figure 5.

Distinct locations and mechanisms underlie regulated Pol II pausing and nucleosome-dependent pausing. (A) Pausing regulated by DSIF and NELF occurs with a predominant peak located 30-nt downstream from the TSS (range = 25–50 nt). This pausing can be long-lived and is clearly modulated to tune gene expression. Release of DSIF/NELF-mediated pausing is triggered by recruitment of P-TEFb to the gene promoter. (B) Upon pause release, Pol II soon encounters the first downstream nucleosome, called the +1 nucleosome. (C) The presence of a nucleosome can transiently halt RNA synthesis and induce Pol II arrest. Association of elongation factors and chromatin modifying complexes greatly facilitate Pol II movement through nucleosomes in vivo, making this pause short lived.