Transcriptional Control by Premature Termination: A Forgotten Mechanism
Authors: Kinga Kamieniarz-Gdula, Nick J. Proudfoot
Journal: Trends in Genetics (2019)

PTT is widespread in metazoans. It can occur close to the TSS or further downstream in the gene body.PTT generates transcripts that, depending on the circumstances, are either rapidly degraded, or are stabilised by polyadenylation, thus contributing to transcriptome diversification.Stable premature transcripts can have independent functions as noncoding (nc)RNA or mRNA encoding proteins with different properties compared with those generated by the full-length transcript.PTT can negatively regulate expression of the full-length transcript and especially controls genes encoding transcriptional regulators.Factors triggering PTT include not only canonical RNA 3′ processing and termination factors, but also other players. Many metazoan factors oppose PTT, thus limiting its damaging potential. The concept of early termination as an important means of transcriptional control has long been established. Even so, its role in metazoan gene expression is underappreciated. Recent technological advances provide novel insights into premature transcription termination (PTT). This process is frequent, widespread, and can occur close to the transcription start site (TSS), or within the gene body. Stable prematurely terminated transcripts contribute to the transcriptome as instances of alternative polyadenylation (APA). Independently of transcript stability and function, premature termination opposes the formation of full-length transcripts, thereby negatively regulating gene expression, especially of transcriptional regulators. Premature termination can be beneficial or harmful, depending on its context. As a result, multiple factors have evolved to control this process. The concept of early termination as an important means of transcriptional control has long been established. Even so, its role in metazoan gene expression is underappreciated. Recent technological advances provide novel insights into premature transcription termination (PTT). This process is frequent, widespread, and can occur close to the transcription start site (TSS), or within the gene body. Stable prematurely terminated transcripts contribute to the transcriptome as instances of alternative polyadenylation (APA). Independently of transcript stability and function, premature termination opposes the formation of full-length transcripts, thereby negatively regulating gene expression, especially of transcriptional regulators. Premature termination can be beneficial or harmful, depending on its context. As a result, multiple factors have evolved to control this process. It is well established that early termination can serve as an important mechanism for transcriptional control. PTT (see Glossary), or 'attenuation', was demonstrated during the mid-1970s to be a key regulatory event for the synthesis of bacterial enzymes that make amino acids [1.Bertrand K. et al.New features of the regulation of the tryptophan operon.Science. 1975; 189: 22-26Crossref PubMed Google Scholar, 2.Artz S.W. Broach J.R. Histidine regulation in Salmonella typhimurium: an activator attenuator model of gene regulation.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3453-3457Crossref PubMed Scopus (43) Google Scholar], and first reported in 1979 to occur for RNA polymerase II (Pol II) transcription of a viral gene in mammalian cells [3.Evans R. et al.Premature termination during adenovirus transcription.Nature. 1979; 278: 367-370Crossref PubMed Google Scholar]. Many more cases of eukaryotic PTT have been identified, even though their analysis was hampered by technical limitations and the highly unstable nature of prematurely terminated RNA. The recent development of next-generation sequencing technologies combined with novel methods to measure nascent transcription, single-molecule footprints, and advanced live-imaging makes it possible to revisit this paradigm. In this review, we present recent findings on metazoan PTT, revealing its widespread nature and role in the regulation of protein-coding genes. While we focus on metazoans, a broader perspective of PTT in other kingdoms of life is summarised in Box 1. Our definition of PTT is the release of Pol II from the gene template between the TSS and 3′-untranslated region (UTR; Figure 1, Key Figure) of the gene. We note that transcription termination is tightly linked with RNA 3′ cleavage and polyadenylation (CPA). Consequently, these two terms are often used ambiguously or even confused in the literature. Multiple recent reviews provide a general background to RNA 3′ processing and transcription termination [4.Di Giammartino D.C. Manley J.L. New links between mRNA polyadenylation and diverse nuclear pathways.Mol. Cell. 2014; 37: 644-649Crossref Scopus (7) Google Scholar, 5.Lemay J-F. Bachand F. Fail-safe transcription termination: because one is never enough.RNA Biol. 2015; 12: 927-932Crossref PubMed Scopus (3) Google Scholar, 6.Libri D. Endless quarrels at the end of genes.Mol. Cell. 2015; 60: 192-194Abstract Full Text Full Text PDF PubMed Google Scholar, 7.Loya T.J. Reines D. Recent advances in understanding transcription termination by RNA polymerase II.F1000Res. 2016; 5: 1478Crossref Google Scholar, 8.Porrua O. Libri D. Transcription termination and the control of the transcriptome: why, where and how to stop.Nat. Rev. Mol. Cell Biol. 2015; 16: 190-202Crossref PubMed Scopus (106) Google Scholar, 9.Porrua O. et al.Transcription termination: variations on common themes.Trends Genet. 2016; 32: 508-522Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 10.Proudfoot N.J. Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut.Science. 2016; 352: aad9926Crossref PubMed Google Scholar, 11.Shi Y. Manley J.L. The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site.Genes Dev. 2015; 29: 889-897Crossref PubMed Scopus (105) Google Scholar] as well as APA [12.Mayr C. Evolution and biological roles of alternative 3′UTRs.Trends Cell Biol. 2016; 26: 227-237Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 13.Neve J. Furger A. Alternative polyadenylation: less than meets the eye?.Biochem. Soc. Trans. 2014; 42: 1190-1195Crossref PubMed Scopus (8) Google Scholar, 14.Tian B. Manley J.L. Alternative polyadenylation of mRNA precursors.Nat. Rev. Mol. Cell Biol. 2017; 18: 18-30Crossref PubMed Scopus (192) Google Scholar, 15.Turner R.E. et al.Alternative polyadenylation in the regulation and dysregulation of gene expression.Semin. Cell Dev. Biol. 2018; 75: 61-69Crossref PubMed Scopus (3) Google Scholar, 16.Zheng D. Tian B. RNA-binding proteins in regulation of alternative cleavage and polyadenylation.Adv. Exp. Med. Biol. 2014; 825: 97-127Crossref PubMed Scopus (24) Google Scholar].Box 1PTT in Bacteria, Yeast and PlantsPTT has been long known to be a key regulatory event in bacteria, referred to as attenuation. Classically, attenuation was shown to control the expression of enzymes involved in amino acid biosynthesis, such as the tryptophan and histidine operons [1.Bertrand K. et al.New features of the regulation of the tryptophan operon.Science. 1975; 189: 22-26Crossref PubMed Google Scholar, 2.Artz S.W. Broach J.R. Histidine regulation in Salmonella typhimurium: an activator attenuator model of gene regulation.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3453-3457Crossref PubMed Scopus (43) Google Scholar]. Bacterial terminators can be intrinsic, associated with a hairpin RNA structure, or factor dependent, usually involving the RNA helicase Rho. Attenuation occurs when an antiterminator hairpin RNA forms ahead of an intrinsic terminator positioned near the 5′ end of an operon. Formation of the antiterminator hairpin precludes the formation of the intrinsic terminator hairpin and so allows transcription to read into the operon and express its protein-coding regions. Switching between the antiterminator and terminator hairpins is controlled by diverse regulators [100.Bastet L. et al.Maestro of regulation: riboswitches orchestrate gene expression at the levels of translation, transcription and mRNA decay.RNA Biol. 2018; 15: 679-682PubMed Google Scholar, 101.Sherwood A.V. Henkin T.M. Riboswitch-mediated gene regulation: novel RNA architectures dictate gene expression responses.Annu. Rev. Microbiol. 2016; 70: 361-374Crossref PubMed Scopus (58) Google Scholar]. Given that translation occurs co-transcriptionally, PTT is closely coupled to translation regulation. This differentiates it from eukaryotic regulation.PTT is also a well-recognised regulatory mechanism in Saccharomyces cerevisiae, mediated by the Nrd1–Nab3–Sen1 (NNS) complex. The first example of attenuation by NNS was demonstrated for the NRD1 gene, which is autoregulated by PTT in response to Nrd1 activity [98.Arigo J.T. et al.Regulation of yeast NRD1 expression by premature transcription termination.Mol. Cell. 2006; 21: 641-651Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. NNS-mediated PTT further regulates genes involved in nucleotide and amino acid biosynthesis, as well as nitrogen metabolism, and is physiologically relevant upon nutritional shift [102.Colin J. et al.Cryptic transcription and early termination in the control of gene expression.Genet. Res. Int. 2011; 2011653494PubMed Google Scholar, 103.Arndt K.M. Reines D. Termination of transcription of short noncoding RNAs by RNA Polymerase II.Annu. Rev. Biochem. 2015; 84: 381-404Crossref PubMed Scopus (25) Google Scholar, 104.Merran J. Corden J.L. Yeast RNA-binding protein Nab3 regulates genes involved in nitrogen metabolism.Mol. Cell. Biol. 2017; 37e00154-17Crossref PubMed Scopus (2) Google Scholar, 105.Bresson S. et al.Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast.Mol. Cell. 2017; 65: 787-800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. The prematurely terminated transcripts sometimes initiate at a TSS upstream of the protein-coding gene [102.Colin J. et al.Cryptic transcription and early termination in the control of gene expression.Genet. Res. Int. 2011; 2011653494PubMed Google Scholar]. Interestingly, it was recently shown that the DNA repair gene DEF1 is attenuated by Sen1 and CPA factors, without Nrd1 and Nab3 involvement [106.Whalen C. et al.RNA polymerase II transcription attenuation at the yeast DNA repair gene, DEF1, involves Sen1-dependent and polyadenylation site-dependent termination.G3 (Bethesda). 2018; 8: 2043-2058Crossref PubMed Scopus (0) Google Scholar]; therefore, PTT in S. cerevisiae might not be limited to the NNS pathway.There are no Nrd1/Nab3 homologues known in plants. However, PTT has an elaborate role in the control of flowering time in Arabidopsis thaliana. FLC is a transcription factor that acts as a master regulator of flowering. It is carefully titrated: small changes in FLC transcript levels significantly affect flowering. The accumulation of FLC mRNA is prevented by FCA and FPA, two RNA-binding proteins associated with RNA 3′-processing factors. FCA and FPA autoregulate their own levels by premature polyadenylation and termination, independently of each other [107.Quesada V. et al.Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.EMBO J. 2003; 22: 3142-3152Crossref PubMed Scopus (200) Google Scholar, 108.Hornyik C. et al.The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.Dev. Cell. 2010; 18: 203-213Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Interestingly, they also promote early termination of the lncRNA COOLAIR [108.Hornyik C. et al.The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.Dev. Cell. 2010; 18: 203-213Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 109.Liu F. et al.Targeted 3′ processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing.Science. 2010; 327: 94-97Crossref PubMed Scopus (227) Google Scholar]. COOLAIR is an antisense transcript to FLC, and functions in early cold-induced silencing of FLC transcription [110.Swiezewski S. et al.Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.Nature. 2009; 462: 799-802Crossref PubMed Scopus (420) Google Scholar]. As a result, several layers of premature termination of coding and noncoding transcripts act to control the timing of plant flowering. It is likely that other examples of PTT will be established in plants. PTT has been long known to be a key regulatory event in bacteria, referred to as attenuation. Classically, attenuation was shown to control the expression of enzymes involved in amino acid biosynthesis, such as the tryptophan and histidine operons [1.Bertrand K. et al.New features of the regulation of the tryptophan operon.Science. 1975; 189: 22-26Crossref PubMed Google Scholar, 2.Artz S.W. Broach J.R. Histidine regulation in Salmonella typhimurium: an activator attenuator model of gene regulation.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3453-3457Crossref PubMed Scopus (43) Google Scholar]. Bacterial terminators can be intrinsic, associated with a hairpin RNA structure, or factor dependent, usually involving the RNA helicase Rho. Attenuation occurs when an antiterminator hairpin RNA forms ahead of an intrinsic terminator positioned near the 5′ end of an operon. Formation of the antiterminator hairpin precludes the formation of the intrinsic terminator hairpin and so allows transcription to read into the operon and express its protein-coding regions. Switching between the antiterminator and terminator hairpins is controlled by diverse regulators [100.Bastet L. et al.Maestro of regulation: riboswitches orchestrate gene expression at the levels of translation, transcription and mRNA decay.RNA Biol. 2018; 15: 679-682PubMed Google Scholar, 101.Sherwood A.V. Henkin T.M. Riboswitch-mediated gene regulation: novel RNA architectures dictate gene expression responses.Annu. Rev. Microbiol. 2016; 70: 361-374Crossref PubMed Scopus (58) Google Scholar]. Given that translation occurs co-transcriptionally, PTT is closely coupled to translation regulation. This differentiates it from eukaryotic regulation. PTT is also a well-recognised regulatory mechanism in Saccharomyces cerevisiae, mediated by the Nrd1–Nab3–Sen1 (NNS) complex. The first example of attenuation by NNS was demonstrated for the NRD1 gene, which is autoregulated by PTT in response to Nrd1 activity [98.Arigo J.T. et al.Regulation of yeast NRD1 expression by premature transcription termination.Mol. Cell. 2006; 21: 641-651Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. NNS-mediated PTT further regulates genes involved in nucleotide and amino acid biosynthesis, as well as nitrogen metabolism, and is physiologically relevant upon nutritional shift [102.Colin J. et al.Cryptic transcription and early termination in the control of gene expression.Genet. Res. Int. 2011; 2011653494PubMed Google Scholar, 103.Arndt K.M. Reines D. Termination of transcription of short noncoding RNAs by RNA Polymerase II.Annu. Rev. Biochem. 2015; 84: 381-404Crossref PubMed Scopus (25) Google Scholar, 104.Merran J. Corden J.L. Yeast RNA-binding protein Nab3 regulates genes involved in nitrogen metabolism.Mol. Cell. Biol. 2017; 37e00154-17Crossref PubMed Scopus (2) Google Scholar, 105.Bresson S. et al.Nuclear RNA decay pathways aid rapid remodeling of gene expression in yeast.Mol. Cell. 2017; 65: 787-800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. The prematurely terminated transcripts sometimes initiate at a TSS upstream of the protein-coding gene [102.Colin J. et al.Cryptic transcription and early termination in the control of gene expression.Genet. Res. Int. 2011; 2011653494PubMed Google Scholar]. Interestingly, it was recently shown that the DNA repair gene DEF1 is attenuated by Sen1 and CPA factors, without Nrd1 and Nab3 involvement [106.Whalen C. et al.RNA polymerase II transcription attenuation at the yeast DNA repair gene, DEF1, involves Sen1-dependent and polyadenylation site-dependent termination.G3 (Bethesda). 2018; 8: 2043-2058Crossref PubMed Scopus (0) Google Scholar]; therefore, PTT in S. cerevisiae might not be limited to the NNS pathway. There are no Nrd1/Nab3 homologues known in plants. However, PTT has an elaborate role in the control of flowering time in Arabidopsis thaliana. FLC is a transcription factor that acts as a master regulator of flowering. It is carefully titrated: small changes in FLC transcript levels significantly affect flowering. The accumulation of FLC mRNA is prevented by FCA and FPA, two RNA-binding proteins associated with RNA 3′-processing factors. FCA and FPA autoregulate their own levels by premature polyadenylation and termination, independently of each other [107.Quesada V. et al.Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.EMBO J. 2003; 22: 3142-3152Crossref PubMed Scopus (200) Google Scholar, 108.Hornyik C. et al.The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.Dev. Cell. 2010; 18: 203-213Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar]. Interestingly, they also promote early termination of the lncRNA COOLAIR [108.Hornyik C. et al.The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.Dev. Cell. 2010; 18: 203-213Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 109.Liu F. et al.Targeted 3′ processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing.Science. 2010; 327: 94-97Crossref PubMed Scopus (227) Google Scholar]. COOLAIR is an antisense transcript to FLC, and functions in early cold-induced silencing of FLC transcription [110.Swiezewski S. et al.Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.Nature. 2009; 462: 799-802Crossref PubMed Scopus (420) Google Scholar]. As a result, several layers of premature termination of coding and noncoding transcripts act to control the timing of plant flowering. It is likely that other examples of PTT will be established in plants. PTT of a protein-coding gene can be divided into termination events occurring close to the TSS or within the gene body (Figure 1). We predict that PTTs at these two locations are likely to be functionally and mechanistically different, since they occur at different stages of the transcription cycle. For many genes, their TSS is characterised by high accumulation of Pol II, as measured by chromatin immunoprecipitation (ChIP). This depends on the action of negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF), typically occurring 30–50 base pairs downstream of the TSS [17.Jonkers I. Lis J.T. Getting up to speed with transcription elongation by RNA polymerase II.Nat. Rev. Mol. Cell Biol. 2015; 16: 167-177Crossref PubMed Scopus (320) Google Scholar]. Such a Pol II 'pileup' is usually interpreted as stable pausing of engaged Pol II. However, it may be also due to PTT with concomitant Pol II turnover [18.Ehrensberger A.H. et al.Mechanistic interpretation of promoter-proximal peaks and RNAPII density maps.Cell. 2013; 154: 713-715Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. Several lines of evidence support the latter: termination and RNA 3′-processing factors have been observed to accumulate at the 5′ ends of genes [19.Brannan K. et al.mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription.Mol. Cell. 2012; 46: 311-324Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 20.Wagschal A. et al.Microprocessor, Setx, Xrn2, and Rrp6 co-operate to induce premature termination of transcription by RNAPII.Cell. 2012; 150: 1147-1157Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar], short nuclear capped transcripts have been detected [21.Nechaev S. et al.Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila.Science. 2010; 327: 335-338Crossref PubMed Scopus (267) Google Scholar], and RNA cleavage sites near the TSS were identified [22.Almada A.E. et al.Promoter directionality is controlled by U1 snRNP and polyadenylation signals.Nature. 2013; 499: 360-363Crossref PubMed Scopus (211) Google Scholar]. Over the past 2 years, more direct experiments have demonstrated that a high percentage of TSS-bound Pol II molecules terminate prematurely. Pol II binding to the genome was recently measured at a single-molecule resolution in Drosophila, with the aid of a novel single-molecule footprinting method [23.Krebs A.R. et al.Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters.Mol. Cell. 2017; 67: 411-422Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. This revealed unexpectedly high levels of Pol II turnover at the promoters of paused genes. In particular, the measured Pol II half-life at promoters of model paused genes was comparable to 'nonpaused', normally elongating genes. Therefore, Pol II accumulation at these promoters appears to be largely due to PTT, rather than to stable pausing of transcription-competent polymerases. This interpretation is further supported by an independent study that analysed the real-time dynamics of Pol II in live human cells using fluorescence recovery after photobleaching (FRAP) [24.Steurer B. et al.Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4368-E4376Crossref PubMed Scopus (0) Google Scholar]. Computational modelling of Pol II kinetics showed that initiating Pol II remains chromatin bound for only 2.4 s and promoter-paused Pol II for 42 s, in contrast to elongating Pol II, which remained chromatin bound on average for 23 min. These big differences in Pol II residence times suggest that only a small fraction of initiating and pausing Pol II proceeds through a complete transcription cycle, whereas most Pol II is released from chromatin at the promoter. Indeed, the determined rate constants showed that only ~10% of Pol II molecules that initiate transcription will go on to promoter pausing and, of those, only ~10% continue into productive elongation. Thus, this study indicates that 99% of transcription initiation events result in PTT at the promoter, with only 1% giving rise to mRNA [24.Steurer B. et al.Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4368-E4376Crossref PubMed Scopus (0) Google Scholar]. This surprisingly inefficient transcription initiation process is consistent with previous Pol II measurements on a lacO array [25.Darzacq X. et al.In vivo dynamics of RNA polymerase II transcription.Nat. Struct. Mol. Biol. 2007; 14: 796-806Crossref PubMed Scopus (0) Google Scholar]. Furthermore, the inhibition of PTT is the most plausible explanation for the dramatic increase in promoter-associated Pol II within 2–3 min after H2O2 addition to U2OS cells [26.Nilson K.A. et al.Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.Nucleic Acids Res. 2017; 45: 11088-11105Crossref PubMed Scopus (15) Google Scholar]. As a further clever way to investigate promoter-associated Pol II, the differential sensitivity of transcription initiation and elongation to high ionic strength has been used [27.Erickson B. et al.Dynamic turnover of paused Pol II complexes at human promoters.Genes Dev. 2018; 32: 1215-1225Crossref PubMed Scopus (9) Google Scholar]. This showed that blocking recruitment of Pol II to promoters (but not elongation) by high salt treatment affected its binding in ChIP followed by next-generation sequencing (ChIP-seq), and revealed an almost complete loss of Pol II from promoter-proximal pause sites within 2–5 min. This loss was rapidly reversible and unaffected by transcriptional inhibitors. Therefore, Pol II removal from pause sites appears not to require elongation. Instead, a high rate of assembly and eviction of pre-initiated Pol II complexes at TSS is predicted. Although the above-mentioned studies used different methodologies, they all describe high turnover rates of Pol II at Drosophila or human promoters in various cell types [23.Krebs A.R. et al.Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters.Mol. Cell. 2017; 67: 411-422Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 24.Steurer B. et al.Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4368-E4376Crossref PubMed Scopus (0) Google Scholar, 25.Darzacq X. et al.In vivo dynamics of RNA polymerase II transcription.Nat. Struct. Mol. Biol. 2007; 14: 796-806Crossref PubMed Scopus (0) Google Scholar, 26.Nilson K.A. et al.Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.Nucleic Acids Res. 2017; 45: 11088-11105Crossref PubMed Scopus (15) Google Scholar, 27.Erickson B. et al.Dynamic turnover of paused Pol II complexes at human promoters.Genes Dev. 2018; 32: 1215-1225Crossref PubMed Scopus (9) Google Scholar]. Therefore, it is unlikely that the observed turnover is an artefact from any one procedure or an unusual cell type. In conclusion, most initiating Pol II molecules appear to terminate prematurely. It follows that the release of Pol II into productive elongation may be regulated by inhibition of this promoter proximal Pol II termination. While promoter-proximal PTT has been largely overlooked, Pol II pausing in this location is well established in metazoans, and is tightly regulated by negative and positive elongation factors, such as P-TEFb [17.Jonkers I. Lis J.T. Getting up to speed with transcription elongation by RNA polymerase II.Nat. Rev. Mol. Cell Biol. 2015; 16: 167-177Crossref PubMed Scopus (320) Google Scholar, 28.Adelman K. Lis J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.Nat. Rev. Genet. 2012; 13: 720-731Crossref PubMed Scopus (533) Google Scholar, 29.Chen F.X. et al.Born to run: control of transcription elongation by RNA polymerase II.Nat. Rev. Mol. Cell Biol. 2018; 19: 464-478Crossref PubMed Scopus (32) Google Scholar]. Several previous studies described longer median half-lives of paused Pol II [30.Henriques T. et al.Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals.Mol. Cell. 2013; 52: 517-528Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 31.Jonkers I. et al.Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.Elife. 2014; 3e02407Crossref PubMed Scopus (215) Google Scholar, 32.Chen F. et al.Stably paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor triptolide.Genes Dev. 2015; 29: 39-47Crossref PubMed Scopus (51) Google Scholar, 33.Shao W. Zeitlinger J. Paused RNA polymerase II inhibits new transcriptional initiation.Nat. Genet. 2017; 49: 1045-1051Crossref PubMed Scopus (46) Google Scholar]. One possible explanation for this discrepancy is the use of triptolide to block initiation. This blocks open complex formation by inhibition of TFIIH-associated XPB [34.Titov D.V. et al.XPB, a subunit of TFIIH, is a target of the natural product triptolide.Nat. Chem. Biol. 2011; 7: 182-188Crossref PubMed Scopus (220) Google Scholar, 35.Alekseev S. et al.Transcription without XPB establishes a unified helicase-independent mechanism of promoter opening in eukaryotic gene expression.Mol. Cell. 2017; 65: 504-514Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar] and was assumed to prevent recruitment of stable Pol II complexes at promoters. However, triptolide also disturbs transcriptional regulation and Pol II stability. Additionally, there is a lag in the onset of XBP inhibition, which may prevent accurate half-life determination [24.Steurer B. et al.Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E4368-E4376Crossref PubMed Scopus (0) Google Scholar, 27.Erickson B. et al.Dynamic turnover of paused Pol II complexes at human promoters.Genes Dev. 2018; 32: 1215-1225Crossref PubMed Scopus (9) Google Scholar]. Further studies using different drugs and methods are required to resolve this discrepancy and so determine the relative contribution of promoter-proximal Pol II pausing versus PTT, to Pol II occupancy at promoters and to the control of productive elongation. Although technically challenging, it will be important to more directly determine the percentage of RNA molecules associated with paused Pol II, which, in physiological conditions, lead to mRNA production; that is, to demonstrate their assumed precursor–product relationship. Notably, paused Pol II blocks transcription initiation of additional polymerases [33.Shao W. Zeitlinger J. Paused RNA polymerase II inhibits new transcriptional initiation.Nat. Genet. 2017; 49: 1045-1051Crossref PubMed Scopus (46) Google Scholar, 36.Gressel S. et al.CDK9-dependent RNA polymerase II pausing controls transcription initiation.Elife. 2017; 6e29736Crossref PubMed Scopus (36) Google Scholar]. As Pol II residence times are variable on different genes [23.Krebs A.R. et al.Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters.Mol. Cell. 2017; 67: 411-422Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 33.Shao W. Zeitlinger J. Paused RNA polymerase II inhibits new transcriptional initiation.Nat. Genet. 2017; 49: 1045-1051Crossref PubMed Scopus (46) Google Scholar] the relevance of P