Getting up to speed with transcription elongation by RNA polymerase II

Abstract

Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.

Figure 1. RNA polymerase II recruitment, initiation and gene entry, pausing and release

a | RNA polymerase II (Pol II) is associated with promoters at, and just downstream of, the transcription start site (TSS). The transcriptional state, position and composition of Pol II are variable and depend on factors that contribute to recruitment, initiation, pausing and release of Pol II. Recruitment of Pol II by general transcription factors (GTFs) results in the formation of a pre-initiation complex (PIC). After rapid Pol II initiation and entry into the pause site, Pol II pausing by negative elongation factor (NELF) and DRB-sensitivity-inducing factor (DSIF) occurs, facilitated by the core promoter elements and the +1 nucleosome. Positive transcription elongation factor-b (P-TEFb) mediates the release of paused Pol II by phosphorylating NELF, DSIF and the carboxy-terminal domain (CTD) of Pol II. DSIF becomes a positive elongation factor after phosphorylation. b | The transcription cycle is predominantly regulated near the TSS, at the steps of recruitment of Pol II to promoters, and release from the promoter-proximal pause site. These steps are most variable in terms of rate (as indicated by the dark blue shading of the boxes defining the steps). Other steps, such as transcription initiation and entry to the pause site, as well as transcription termination from the pause site, seem not to be as variable in rate and less subject to regulation (as indicated by the lighter blue shading of the boxes).

Figure 2. The cascade of events that precede productive elongation by RNA polymerase II

The regulatory steps in the transcription cycle are mediated by a large number of factors, cofactors and regulatory mechanisms that cooperatively mediate the recruitment of RNA polymerase II (Pol II) and release of paused Pol II at both enhancer regions and gene start sites. a | Inactive transcription start sites (TSSs) at enhancers and genes are often occupied by nucleosomes. b | Nucleosomes are pushed aside (indicated by arrows) in response to external signalling events and the recruitment of transcription factors (TFs) that work with nucleosome remodellers and modifiers. c | Upon binding of the general transcription machinery at the TSSs, Pol II is recruited, initiated and phosphorylated at Ser5, which promotes transcription initiation. Subsequently, Pol II advances to the pause site, where it is stabilized by pausing factors (shown in red), such as negative elongation factor (NELF) and DRB-sensitivity-inducing factor (DSIF). d | Further signalling events lead to the recruitment of additional TFs, such as nuclear factor-κB (NF-κB) or MYC, which can recruit positive transcription elongation factor-b (P-TEFb) directly (not shown), or indirectly via co-activators such as bromodomain-containing protein 4 (BRD4). e | Finally, the cumulative recruitment of TFs results in binding of co-activators like BRD4, attracts other co-activators and elongation factors (shown in different shades of blue) such as the super elongation complex (SEC) and Mediator, potentially causing enhancer–promoter interactions in cooperation with non-coding RNAs (ncRNAs). This results in the activation of P-TEFb, which phosphorylates pausing factors and the carboxy-terminal domain (CTD) of Pol II, leading to Pol II pause release and progression into a phase of productive elongation. This results in mRNA synthesis at the gene, and production of enhancer RNAs (eRNAs) at the enhancer (not shown).

Figure 3. RNA polymerase II elongation rate throughout a gene

a | The density of RNA polymerase II (Pol II) and the elongation rates it achieves are not constant, but can vary throughout the gene, as exemplified by a schematic representation of a hypothetical gene. The changes in density of engaged Pol II, and the relative Pol II elongation rates, are indicated on the y-axis and the green shading. Pol II is slowest at the promoter-proximal pause site, and speeds up after release from the pause site over a region of approximately 15kb. Further variations in elongation rates are measured around exons (blue bars), where Pol II slows down, and at the site of termination, where Pol II pauses to enable efficient termination. Factors that affect the rate of transcription by Pol II are listed. Slowdown around exons might be orchestrated by a combination of CG content, nucleosome occupancy, histone marks and splicing factors that are enriched at exons. Pausing of Pol II near the polyadenylation site at the 3′ ends of genes could be caused by a combination of R-loops, histone marks and termination factors. b | Elongation rates can be measured by determining nascent transcription after a block of release of new Pol II into productive elongation in a timed manner; the distance travelled by the ‘retreating wave’ of Pol II that is already productively elongating within a period of time is indicative of the elongation rate. c | Elongation rates can also be measured by determining nascent transcription after wash-out of inhibitory drugs; the distance travelled by the ‘emerging wave’ of newly released productive Pol II within a period of time is indicative of the elongation rate.