RNA polymerase II elongation factors use conserved regulatory mechanisms

RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.


Introduction
Transcription is the first step in gene expression and underlies development and cell function.Misregulation of transcription is related to cancerous cell growth and disease [1e3].Therefore, interfering with the regulation of transcription has therapeutical potential [4].Transcription of mRNAs is carried out by RNA polymerase II (Pol II) and regulated during initiation, elongation and termination, which require initiation, elongation and termination factors [5].During initiation, Pol II and general transcription factors assemble on promoter DNA, forming a preinitiation complex, followed by DNA opening and initial RNA synthesis, resulting in an initial transcribing complex (ITC), as reviewed in Refs.[5,6].Pol II then escapes from the promoter and forms a stable elongation complex.

Regulation of Pol II elongation by the PAF complex
Human PAF consists of PAF1, LEO1, CTR9, CDC73, WDR61 and RTF1 [1,9].RTF1 was shown to stimulate Pol II transcription in nuclear extracts [26].Cryo-EM structures of the Pol II-DSIF-SPT6-PAF complexes showed that PAF has at least two functions in vitro.First, it promotes the release of Pol II from the paused elongation complex by competing with NELF for Pol II binding at Pol II foot domain [9].Second, PAF-RTF1 directly stimulates the activity of Pol II and the presence of PAF and a RTF1 'latch' element is required for the stimulation [10].
In the structure, the PAF1-LEO1 subcomplex contacts Pol II external 2 and protrusion domains via a TFIIFlike triple b-barrel structure (dimerization domain) and a LEO1 C-terminal extension, respectively (Figure 2a, b).RTF1 further locks PAF1-LEO1 onto the Pol II surface and extends the RTF1 'latch' to a groove formed by the Pol II funnel helices and the bridge helix [10] (Figure 2c).This region is also the binding site for a loop insertion in yeast Pol II Rpb2 (Figure 2c).Interestingly, the transcription elongation rate of yeast Pol II is about 5 times faster than mammalian Pol II [29].The presence of the RTF1 latch may allosterically regulate the activity of Pol II by changing the dynamics of the bridge helix, a mobile element of the Pol II active center [10].
The super elongation complex promotes the release of promoter-proximal paused Pol II by delivering P-TEFb [1,9] and also stimulates transcription elongation via its ELL-EAF subcomplex in vitro [21,29,31,33].It regulates elongation of genes that encode heat shock and other stress response molecules, developmental regulators and enhancer RNAs [18].The little elongation complex regulates the transcription cycle of snRNAs and snoR-NAs [18,40].Despite the importance of SEC and LEC in transcription regulation, how SEC and LEC interact with Pol II and stimulate elongation was unclear.
The recent cryo-EM structure of the Pol II-ELL2-EAF1 complex partially provides an answer to this question [41].In this structure, ELL2-EAF1 binds to the Pol II lobe, external 2 and protrusion domains via their Nterminal dimerization domains, an ELL2 helix and a linker extension (Figure 2a, b).The ELL2-EAF1 dimerization domains fold into TFIIF-like triple bbarrel structure and occupy the same binding site as TFIIF (Figure 2b, 3a, b).Biochemical assays further show that the ELL2 protrusion-interacting linker is required for the elongation stimulation activity of ELL2.Structural analysis shows that binding of ELL2-EAF1 induces conformational change at Pol II lobe domain, resulting in a narrower DNA cleft (Figure 3c).We propose that the binding of ELL2-EAF1 complex allosterically stimulates transcription elongation.This study indicates that SEC could bind to Pol II via the ELL-EAF subcomplex and exert its function.As LEC also contains the ELL-EAF subcomplex, it is likely that LEC could bind to Pol II via ELL-EAF as well, although it is not clear whether other subunits of LEC also interact with Pol II.However, considering that SEC and LEC regulate different genes, additional mechanism must exist that render them gene-specific.Future structural studies involving more subunits and binding partners of LEC and SEC can provide further insights.
In the Pol II-SPT6-Elongin structure, Elongin interacts with the Pol II domains external 2 and protrusion via the helix a5 of the ELOA superfamily domain and an ELOA C-terminal linker, respectively (Figure 2a, b).Additionally, an ELOA 'latch' element binds near the Pol II funnel helices, in the same region as that of the RTF1 latch and the yeast Rpb2 insertion (Figure 2c).Biochemical assays show that the ELOA latch and Cterminal linker are required for the elongation stimulation activity of ELOA.Importantly, binding of Elongin induces a conformational change at the Pol II funnel helices and the ELOA latch is required for this conformational change (Figure 3d).We propose that Elongin allosterically regulates Pol II activity, probably by promoting translocation or stabilizing Pol II at a more active conformation.
Elongin also interacts with the Cullin-RING E3 ubiquitin ligase CUL5-RBX2 complex, forming a pentameric ubiquitin ligase complex, under oxidative or other stresses or upon UV irradiation [18,43,44].This pentameric Elongin complex promotes degradation of the RPB1 subunit of stalled Pol II [45,46].The structure of the Pol II in complex with trimeric Elongin indicates that the 5-subunit Elongin could bind to Pol II via ELOA.However, whether there is an additional contact between Pol II and the 5-subunit Elongin ubiquitin ligase is unknown.How the 5-subunit Elongin ubiquitylates Pol II also remains to be investigated.

General mechanisms for elongation stimulation
Comparison of the structures of Pol II in complex with PAF-RTF1, Elongin, ELL-EAF and TFIIF reveals a general mechanism that regulates transcription elongation.It appears that at least three tethering sites between elongation factors and Pol II are required for their stimulatory transcription elongation activity.Pol II protrusion and external 2 domains are the two common binding sites for these elongation factors.The third binding site is at the Pol II lobe or funnel helices, which varies depending on the factors (Figure 3a 3c, d).In the case of ELL2-EAF1, the Pol II lobe domain moves towards the DNA cleft, forming a more compact cleft (Figure 3c, d, middle panel).In the case of Elongin, the funnel helices move towards the trigger loop, which might promote

Factor exchange during the transition from initiation to elongation
The exact mechanism for the transition from initiation to elongation is not entirely clear.Possible mechanisms are replacement of TFIIB by extending RNA at a certain length and competition between initiation factors and elongation factors for Pol II binding, based on structural comparisons between initiation complexes and the paused elongation complex [6].Unlike other initiation factors, TFIIF may associate with early elongating Pol II and function as an elongation factor [18].The overlap between the binding site of PAF, ELL and Elongin, and TFIIF on Pol II indicates that these factors cannot bind Pol II simultaneously (Figure 3a).This suggests that replacement of TFIIF by these elongation factors contributes to the transition from initiation to elongation.What determines which factor replaces TFIIF for a certain gene is unclear.PAF, Elongin and SEC appear to have distinct functions in vivo [18], indicating that they could regulate different sets of genes.The mechanism underlying the target selection of the elongation factors is of interest for future studies.
The PAF1-LEO1 in the EC* complex was shown to compete with the INTS1 subunit of Integrator [11].Similarly, TFIIF, ELL-EAF and Elongin also clash with Integrator at the external 2 and protrusion domains.This suggests that they could also prevent pre-mature termination during processive elongation.What is intriguing is that LEC, which contains ELL-EAF as a subcomplex, recruits the Integrator complex to snRNAs [39].How the two factors may co-exist in one complex requires further investigation.to Pol II simultaneously.Superposing the structures of the elongation complexes containing TFIIF, ELL2-EAF1 or Elongin onto the TFIIS containing Pol II elongation complexes also showed no physical clash between these elongation factors and TFIIS (Figure 4a,  b).This may suggest that TFIIF, ELL and Elongin might change the accessibility of the 3 0 -end of the RNA for TFIIS.

Conclusions and future research directions
In summary, cryo-EM analysis enabled the determination of many structures of Pol II elongation complexes with bound elongation factors.This greatly increased our understanding of the regulation of transcription elongation.However, the new results also raise new questions.First, how do elongation factors cooperate with each other?Despite the overlapping binding site on the Pol II surface, Elongin was shown to interact with PAF and the Integrator complex [50,51] and LEC interacts with Integrator subunits [40].There seems to be an intricate functional network among elongation factors in vivo.Dissecting this interaction network structurally will advance our understanding of elongation regulation.Second, how do SEC, LEC and Elongin define their target elongation complexes?PAF seems to be a general elongation factor for pause release, but Elongin also functions in pause release.SEC and LEC share the same ELL-EAF interface with Pol II, but they regulate different sets of genes.It would be interesting to study how additional factors facilitate elongation factor recruitment.Third, how does the 5-subunit Elongin ubiquitin ligase ubiquitylate Pol II? Elongin and the 5-subunit Elongin ubiquitin ligase share the same ELOA-Pol II interaction interface, however, their functions differ.What is the mechanism that induces the switch of Elongin function from an elongation factor to a ubiquitin ligase?Fourth, how do SEC, LEC and Elongin function in the context of chromatin and in response to cellular signals and stress?Answering these questions would advance our understanding of transcription regulation.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figure 1 Overview
Figure 1

Figure 2 Structures
Figure 2 , b).First, TFIIF, PAF1-LEO1, ELL-EAF and Elongin bind to the external 2 domain of Pol II RPB2.Second, a C-terminal linker interacts with the protrusion domain of Pol II.Third, RTF1 and Elongin, but not TFIIF and ELL-EAF, contain a latch that interacts with the funnel helices of Pol II.On the other hand, TFIIF and ELL-EAF contain a triple b-barrel structure that interacts with Pol II lobe domain.The three tethering sites potentially synergize to stabilize an active Pol II conformation.The conformational changes in Pol II upon binding of elongation factors are different, depending on the elongation factors bound (Figure

Figure 3 Comparison
Figure 3 Repression of TFIIS-stimulated cleavage of nonarrested Pol II transcripts by TFIIF, Elongin and ELL TFIIS is the first elongation factor identified and increases the Pol II elongation rate [47].During elongation, Pol II translocates and sometimes backtracks.Extensive backtracking leads to arrest of Pol II and extrusion of the 3 0 -end of the RNA into a pore beneath the active site [48].TFIIS rescues backtracked Pol II by stimulating the endonuclease activity of Pol II [18].After cleavage, the 3 0 -end of RNA is re-aligned in the active site [17].Elongation factors TFIIF, ELL and Elongin were shown to repress transient pausing and TFIIS stimulated cleavage of transiently paused but not stalled Pol II transcripts [25].However, it is unclear whether elongation factors counteract the activity of TFIIS by competitive binding to Pol II or by changing the accessibility of TFIIS to 3 0 -end of the RNA transcript.As revealed by the structure of the Pol II-DSIF-PAF-SPT6-TFIIS-nucleosome structure [48], PAF and TFIIS bind

Figure 4 ELL
Figure 4 47. Sekimizu K, Kobayashi N, Mizuno D, Natori S: Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II.Biochemistry 1976, 15: 5064-5070.48.Gonzalez MN, Blears D, Svejstrup JQ: Causes and consequences of RNA polymerase II stalling during transcript elongation.Nat Rev Mol Cell Biol 2021, 22:3-21.49.Filipovski M, Soffers JHM, Vos SM, Farnung L: Structural basis of nucleosome retention during transcription elongation.Science 2022, 376:1313-1316.50 * * .Wang YT, Hou LM, Ardehali MB, Kingston RE, Dynlacht BD: Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity.J Biol Chem 2021:296.This paper describes the role of the Elongin A subunit of Elongin during transcritpion in vivo.The results suggest that Elongin may regulate pause release of Pol II and identified its interaction partners.51 * * .Ardehali MB, Damle M, Perea-Resa C, Blower MD, Kingston RE: Elongin A associates with actively transcribed genes and modulates enhancer RNA levels with limited impact on transcription elongation rate in vivo.J Biol Chem 2021:296.This paper describes the role of the Elongin A during transcritpion in vivo.This study suggests that Elongin A regulates the transcription of ehancer RNAs and identified interaction partners of Elongin A in vivo.52.He Y, Yan C, Fang J, Inouye C, Tjian R, Ivanov I, Nogales E: Near-atomic resolution visualization of human transcription promoter opening.Nature 2016, 533:359-365.