Widespread peaks of elongating Pol II at 20 to 100 nt near promoters in eukaryotes were initially described for several model genes, including the mammalian β-globin locus233, Drosophila heat shock genes 234, human c-myc and c-fos genes 235-238. Over the last decades 239,240, promoter-proximal pausing of Pol II has emerged as a general phenomenon across protein-coding genes (including up to 90 % of active promoters 241), and has blossomed into a rich research field. More recently, Pol II pausing was reported at noncoding genes that produce long noncoding RNAs 242,243, including enhancer RNAs 182, and upstream antisense RNAs 244. In this chapter, I briefly summarize the biological impact of Pol II pausing in human cells, its regulation – focusing on the prominent release factor P-TEFb –, and an emerging model of the molecular mechanism of Pol II pausing.
What biological functions might Pol II pausing serve in human cells? Interestingly, Pol II pausing occurs at both, genes actively producing RNA and genes without efficient transcript completion 19. At genes that are fully transcribed, pausing facilitates the assembly of RNA processing factors 245. This pausing checkpoint ensures that 5’ ends of nascent RNA molecules are capped and protected from degradation prior to elongation 246,247. Genes that experience initiation but not elongation are often in an uninduced state 248. As a result, the Pol II pausing associated transcription machinery alters chromatin and maintains an open promoter structure enabling regulatory factors to access underlying DNA elements 18. Upon developmental regulation 249,250, or in response to other stimuli 251,252 pausing is reduced and responsive genes are fully transcribed. Furthermore, pausing was shown to synchronize gene activation events in Drosophila 253. Negative consequences of pausing may be transcription-replication conflicts during S phase, especially when paused Pol II encounters the transcription-replication machinery head-to-head, leading to DNA damage and genomic instability 254. Paused Pol II could also interfere with transcription of other genes in its vicinity by steric hindrance 255. What factors affect Pol II pausing in human cells? At the outset of my graduate work, it has been reported in Drosophila 256 and Escherichia coli257 that the DNA sequence composition is affecting Pol II pausing. However, the underlying sequence determinants at human genes were unknown. Similar to other transcription steps (section 1.1), pausing is stabilized by several factors, including DSIF composed of Spt4 and Spt5 258 and the NELF complex with A, B, C/D and E subunits 259. Paused Pol II has been shown to relocate nucleosomes 260, and vice versa, nucleosomes seem to enhance pausing 261. Additional factors influence the stability of paused Pol II, such as GDOWN1 and TFIIF 262. Most prominent among the pause release factors is the CDK9-containing kinase complex P-TEFb 263-265. Other factors involved in the transition to productive elongation are the PAF1 complex 110,266-268, the elongation factors SPT6
269-272, and TFIIS 273, as well as several other factors recruiting P-TEFb.
2.1 Positive transcription elongation factor b (P-TEFb)
The positive transcription elongation factor b (P-TEFb) 263 is a heterodimer which is constitutively expressed throughout the cell cycle 274 (recently reviewed in 275,276). It consists of the cyclin-dependent kinase CDK9 originally termed PITALRE 264,277 and a T-type cyclin (CCNT1 or CCNT2) 278-280. In human cells, P-TEFb is either active or inactive 281,282. In its inactive form, 7SK RNA serves as a scaffold for interacting proteins (LARP7, MePCE, HEXIM1, HEXIM2 283), ultimately sequestering P-TEFb in a 7SK small nuclear ribonucleoprotein complex (snRNP) 284. The ratio of both forms varies between different cell lines but the majority is inactive in steady state 281,282,285.
Recruitment and activation of P-TEFb. Both forms of P-TEFb can be recruited to its target genes via several different recruitment complexes including (co-)activators or chromatin-associated factors (reviewed in 276). Activators as C-MYC 240,286, NF-kappaB 287 or the viral HIV Tat 288 transcription factors can directly recruit active P-TEFb to target genes by physically interacting with its CCNT subunit. The subunit MED26 of the Mediator complex binds the super elongation complex (SEC) which contains active P-TEFb, ELL/EAF family members and other factors 289. Another recruitment mechanism of active P-TEFb utilizes histone tail binders such as BRD4 or MePCE. BRD4 interacts with promoter-proximal histone 4 acetylated lysine (H4K16ac) via its bromodomain 290,291. MePCE acts independently during P-TEFb recruitment by interacting with histone H4 of the +1 nucleosome 292. The inactive, 7SK snRNP-bound P-TEFb can be nuclear or chromatin-bound by TRIM28/KAP1 293. Nuclear 7SK snRNP-bound P-TEFb is activated by T-loop phosphorylation of CDK9 by CDK7 which frees P-TEFb from the 7SK snRNP complex 294,295. However, it is not known which factor releases P-TEFb from the chromatin-associated 7SK snRNP complexes. To date, our understanding of P-TEFb recruitment remains incomplete and needs to be established at a genome-wide scale. The timing of P-TEFb activation and the localization of its recruitment complexes at a certain gene might define the length of the pause duration.
P-TEFb controlled checkpoints: promoter-proximal pausing and pA site. After recruitment to its target and activation, the CDK9 kinase phosphorylates the Spt5 subunit of DSIF 296, NELF
297 and the CTD of POLR2A 298. For the latter, CDK9 has been shown to phosphorylate the POLR2A linker region of the CTD 110,111, as well as serine 2 299,300 and serine 5 301 of the CTD heptad repeats (Figure 1 c). CDK9 was recently shown to be implicated in a second checkpoint around the polyadenylation site 302. The second checkpoint is still poorly understood and might implicate CDK9 in 3’-end RNA processing and transcription termination 302,303. This implication is supported by the observation that P-TEFb enhances the activity of the transcription termination factor XRN2 by phosphorylation 303.
2.2 Integration of pausing models
Today we have a better knowledge of the biochemical composition of paused and elongating Pol II complexes, and many pause and release factors have been identified 304. But our knowledge about the timing and molecular mechanisms of assembly and composition of Pol II
establishing promoter-proximal pausing were described in the literature: the kinetic model, the barrier model and the interaction model (reviewed in 304). The kinetic model describes pausing as a combination of the slow elongation rate of Pol II (see section 3.1.1), reversible sliding of Pol II with low processivity along DNA and RNA (backtracking) 305 and the delayed recruitment rate of Pol II release (P-TEFb complex) and elongation factors prior to productive elongation. The barrier model (also referred to as ‘ubiquitous pausing’ 46) builds on observations that nucleosomes, especially the first downstream of the TSS, hinder Pol II’s transition to productive elongation 261. The interaction model suggests that factors stabilizing the paused Pol II complex such as NELF, DSIF, or other factors (of which the activity still needs to be established) determine pausing. Depending on the gene’s architecture and nanoenvironment, the energy landscape and thus, the transition rate to productive elongation will be variable and each of the three models might contribute differently to establishing a paused Pol II 306 (see also Supplementary Note 1).
Recent studies revealed structures of the Pol II elongation complex in the paused and activated state, and provided the first mechanistic insights into the P-TEFb dependent switch to active elongation 110,307 (Figure 2 a). The RNA-DNA hybrid within the paused polymerase is in the tilted state that hinders nucleotide addition at the active site 307. Thus, the subsequent nucleotide is not added yet. We defined the pause site (position 0) to be the position in line with the 'post-translocated' RNA rather than with the 'pre-translocated' DNA (see Methods II.2.2.4). A tilted hybrid might be the hallmark of a paused state and was also observed in bacterial elongation complexes 308.
Figure 2. Structural modeling of promoter-proximal pausing and initiation.
(a) Top: close-up view of the DNA-RNA hybrid in the paused transcription elongation complex (paused EC) (in dark blue and red) (PDB-code 6GML 307) compared to the elongation complex (EC) (in silver) (PDB-code 5OIK 105). In the paused EC, the DNA-RNA hybrid is in an offline transcription state. The template DNA strand (in dark blue) passes over the Pol II bridge helix (in green). Structural view (top) was kindly provided by Dr. Seychelle Vos (MPI-bpc, Dept. of Molecular Biology) 307. Bottom: schematic of nucleic acid residues (DNA in blue, RNA in red). Shaded area highlights the DNA-RNA hybrid. The pause site (n*) (throughout this work referred to as ‘position 0’) is denoted at +1 of the template DNA. Bottom schematic is adapted from Armache et al. 309. (b) Modeling shows that paused Pol II (silver, right) positioned 50 bp downstream of the TSS allows for formation of the Pol II initiation complex (different colors, left). Modeling is based on the latest structural information (Mediator EMD-8307 310, TFIID EMD-3305 311, TFIIH EMD-3307 312, closed complex PDB-code 5FZ5 313, EC PDB-code 1WCM 314). Structural modeling was performed by Dr. Merle Hantsche (MPI-bpc, Department of Molecular Biology).
Jesper Svejstrup and co-workers explored footprints of initiating and paused Pol II molecules and proposed a theoretic model (referred to as ‘Ehrensberger theory’) in which a paused polymerase interferes with the binding of the Pol II initiation complex, or a newly initiating polymerase triggers the release of a paused polymerase to productive elongation 315. As a consequence, the authors suggest that initiation might still be the rate limiting step of transcription and pausing might serve as a window of opportunity to collect necessary transcription elongation factors for full processivity during elongation 315. Structural modeling shows that Pol II positioned 50 bp downstream of the TSS allows for formation of the Pol II initiation complex while shorter distances between the active sites of paused and initiating Pol II are predicted to lead to steric clashes (Figure 2 b). Even if a paused Pol II is located further downstream, it may still restrict initiation events if additional polymerases line up behind it.