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. 2026 May 7;9:623. doi: 10.1038/s42003-026-10203-1

RNA polymerase II transcription initiation with and without XPB translocase activity

Mario Zurita 1,✉, Samantha Cruz-Ruiz 1, Eduardo Calvario 1
PMCID: PMC13153155  PMID: 42098258

Abstract

A key step in gene expression is the opening of DNA at the transcription start site (TSS), allowing RNA polymerase to begin RNA synthesis. In eukaryotes, RNA polymerase II initiates transcription within the pre-initiation complex (PIC), which includes TFIIH. The XPB subunit of TFIIH drives DNA unwinding at the TSS through ATP-dependent activity. However, some promoters enable transcription without XPB, likely due to intrinsically unstable DNA that does not require active unwinding. This mechanism may reflect an evolutionary remnant of archaeal transcription.

Subject terms: Transcriptional regulatory elements, Gene regulation

This perspective suggests that transcription initiation at certain human promoters does not require the translocase activity of the XPB subunit of TFIIH, as their intrinsic thermodynamic instability enables DNA opening without active unwinding.

TFIIH is a versatile, multifunctional protein complex

During evolution, many cellular factors have been selected for their ability to perform multiple functions. This multifunctionality enables communication between distinct cellular processes, such as different stages of the cell cycle or responses to stress. In eukaryotes, one well-studied example is TFIIH, a complex involved in both transcription and DNA repair. TFIIH is composed of ten subunits organized into two subcomplexes: the core and the Cyclin-Activating Kinase (CAK) module. The core consists of seven proteins, while CAK is composed of three. Whereas the TFIIH core is essential for Nucleotide Excision Repair (NER), the CAK functions independently in cell cycle regulation, and both subcomplexes together are required for transcription initiation by RNA Pol II. In humans, the core includes XPB/ERCC3, XPD/ERCC2, p62/GTF2H1, p52/GTF2H4, p44/GTF2H2, p34/GTF2H3, and p8/GTF2H5. The CAK subcomplex contains CDK7, cyclin H, and MAT1. Transcriptional activity of TFIIH requires the assembly of both subcomplexes into the holo-TFIIH complex1,2.

Several TFIIH subunits possess enzymatic activities. XPD is an ATP-dependent helicase that unwinds DNA in a 5′ to 3′ direction, while XPB functions as an ATP-dependent translocase that facilitates DNA opening at promoter regions, an activity considered critical for transcription initiation by RNA Pol II. In parallel, CDK7 phosphorylates other CDKs to regulate the cell cycle and the C-terminal domain (CTD) of RNA Pol II, promoting transcription initiation3,4. Beyond these functions, TFIIH has been implicated in additional cellular processes, including telomere maintenance, chromosome condensation, and chromosome segregation during mitosis5–9.

Although TFIIH was discovered over three decades ago, it remains the focus of ongoing research due to its diverse cellular roles. Numerous reviews have addressed the many functions of TFIIH2,10. In this Perspective, we focus specifically on the XPB subunit and its role in transcription initiation, as XPB is essential yet intriguingly appears to be dispensable for the transcription of certain genes.

The role of XPB in transcription initiation

XPB is a multifunctional enzyme with several domains. Its helicase-ATPase core, formed by two RecA-like domains (HD1 and HD2), drives ATP-dependent DNA translocation at promoters (Fig. 1a). The N-terminal domain interacts with the p52 and p8 subunits of TFIIH to regulate XPB activity (Fig. 1b), while the Damage Recognition Domain (DRD) and RED motif stabilize TFIIH at DNA damage sites and confine XPB function to the assembled complex. The Thumb-like (ThM) motif supports DNA positioning and promoter opening, and the C-terminal domain is required for TFIIH assembly1,2 (Fig. 1c).

Fig. 1. Structural perspectives on human XPB and TFIIH.

Fig. 1

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a Domain organization of human XPB with its predicted structure generated by AlphaFold 357. b XPB-p52-p8 submodule predicted with AlphaFold 3. The predicted structures are similar with the structures reported using X-Ray diffraction and High Resolution Cryo-Microscopy. p52 and p8 modulate the ATPase activity of XPB. p52 together with the small subunit p8, forms a ring-like clamp around XPB’s helicase domains, reducing flexibility and stabilizing a conformation suitable for ATP hydrolysis. c The human holo-TFIIH complex predicted with AlphaFold 3 and a model of its configuration. AlphaFold 3 was also used to visualize intrinsically disordered regions, which are typically not resolved by X-ray crystallography or cryo-electron microscopy. d Structure of the human holo-PIC in the closed state (PDB 8BVW) and a structural model of the PIC interacting with the +1 nucleosome (PDB: 8BVW). Inside the PIC, TFIIH directly contacts the RNA Pol II and TFIIE. In addition, TFIIH directly contacts the +1 nucleosome, with the XPB translocase inducing DNA twisting and partial unwrapping of nucleosomal DNA. e Schematic representation of the pre-initiation complex (PIC) illustrating the XPB-mediated DNA translocase activity essential for promoter opening and transcription initiation. Arrows denote XPB-driven DNA translocation and the force exerted to move the downstream nucleosome.

RNA Pol II cannot recognize promoters or initiate transcription in a regulated manner on its own; it requires the assistance of the general transcription machinery. During transcription initiation, transcription factors bound to proximal or distal regulatory elements recruit the components necessary to assemble the PIC (Fig. 1d)11. The process begins with TFIID, which contains the TATA-binding protein (TBP), recognizing core promoter elements. TFIIA and TFIIB then bind upstream of the TSS and facilitate the recruitment of RNA Pol II in complex with TFIIF11. Subsequently, TFIIE binds to the complex and enables the recruitment of TFIIH, completing the formation of the PIC (Fig. 1d)12–18. Once assembled, the PIC is further stabilized and modulated by the Mediator complex, which in human cells consists of 25-30 subunits and plays a crucial role in modulating the activation of transcription initiation15,16,19. In addition, gene-specific transcription factors influence the assembly and recruitment of various PIC components12.

Following PIC assembly, the DNA around the TSS must be unwound, transitioning the complex from a closed to an open state. High-resolution cryo-electron microscopy has revealed the three-dimensional structure of the human and yeast PICs in several functional states, including the closed complex, open complex, and the initiating complex13. Remarkably, the PIC structure has also been resolved in the presence of the +1 nucleosome20–22.

These structural studies show that both TFIID and Mediator help stabilize TFIIH. Within the PIC, CDK7 (part of the CAK subcomplex of TFIIH) is positioned to phosphorylate Ser5 of the heptapeptide repeat in the CTD of RNA Pol II, while XPB is localized near the TSS to mediate promoter DNA opening15,16. DNA unwinding at the TSS is primarily driven by TFIID and TFIIH, with XPB’s translocase activity playing a central role23. When Mediator interacts with the TFIIH core, it positions the two helicase domains of XPB to bind downstream of the promoter. This enables XPB to translocate DNA toward RNA Pol II, promoting strand separation both on naked DNA templates and in chromatin contexts where the PIC is assembled near the +1 nucleosome (Fig. 1d, e)20–22. However, when the +1 nucleosome is located fewer than 10 base pairs downstream of the TSS, TFIIH tends to adopt a closed conformation and only partially engages with the DNA, potentially limiting XPB’s ability to promote DNA opening20. Importantly, the +1 nucleosome often contains histone variants H2A.Z and H3.3, and H3 frequently exhibits acetylation at lysine 27 (H3K27ac), making the nucleosome inherently unstable21. It has been proposed that nucleosomes located within ~10 bp of the TSS may sterically hinder XPB function, while those positioned farther downstream allow TFIIH to adopt an open conformation, facilitating promoter melting. In human cells, some TFIIH subunits directly interact with the +1 nucleosome. For instance, p52 contacts the H2A.Z-H2B dimer, and XPB engages with nucleosomal DNA. This interaction appears dynamic and may facilitate repositioning of the nucleosome away from the PIC21. Indeed, it has been proposed that TFIIH, and in particular XPB, may function as a chromatin remodeler. Through its ATP-dependent translocase activity, XPB can push downstream DNA toward the PIC, shifting the +1 nucleosome and thereby enabling efficient transcription initiation21,24.

Nevertheless, not all promoters require XPB’s ATPase activity for transcription to occur. Some promoters can initiate transcription efficiently even in the absence of XPB´s translocase function (discussed below), suggesting that the requirement for XPB may be context-dependent and influenced by chromatin architecture.

XPB in archaea: present but not required for transcription initiation

Functional orthologues of the two TFIIH helicases, XPB and XPD, are present in Archaea, although the remaining subunits that constitute the eukaryotic TFIIH complex are absent. In Archaea, research on XPB and XPD has primarily focused on their roles in NER25. For instance, archaeal XPB has been shown to bind the endonuclease Bax1, forming a complex that participates in DNA repair26. XPD, on the other hand, appears to function exclusively in NER, and there are no reports demonstrating an interaction between archaeal XPB and XPD. The archaeal XPB contains an ATPase catalytic domain and retains ATPase activity (Fig. 2a)25.

Fig. 2. Structural and schematic representations of archaeal XPB and promoter.

Fig. 2

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a Domain organization of archaeal XPB, including its predicted structure, and comparison with human XPB. An overlap of the two structures is also shown. b Schematic representation of the archaeal promoter and general factors that participate in transcription initiation. AfTFB, the archaeal homolog of TFIIB, comprises four structural modules: an N-terminal region involved in TSS determination, central cyclin-fold domains that interact with AfTBP and RNA polymerase, an elongated α-helical region that facilitates the transition to elongation, and a C-terminal α-helical domain that stabilizes TBP-DNA interactions. AfTBP is highly conserved and adopts a saddle-shaped fold that binds the DNA minor groove, induces DNA bending, and promotes recruitment of TFB and RNA polymerase. AfTFE contains disordered N- and C-termini flanking a central helix-turn-helix motif and functions to stabilize the transcription initiation bubble. Structures in a and b were modeled with AlphaFold 357 from Archaeoglobus fulgidus (Af) protein sequences.

The potential role of XPB in transcription within Archaea remains under debate. Archaeal RNA polymerase is highly homologous to eukaryotic RNA Pol II, sharing all 12 subunits including the large catalytic subunit of RNA Pol II, which in eukaryotes contains the CTD with its characteristic heptapeptide repeats. Similar to the eukaryotic system, archaeal transcription requires general factors functionally analogous to TBP, TFIIB (TFB in Archaea), and TFIIE (TFE; Fig. 2b)27,28. During transcription initiation, archaeal RNA polymerase depends on TFE, which binds the clamp and stalk regions of RNA polymerase and helps stabilize the transcription bubble29. As mentioned before, in eukaryotes, TFIIE recruits TFIIH and stimulates XPB’s translocase activity17.

Interestingly, in some archaeal species, TFE has been shown to interact with XPB, suggesting a possible mechanism analogous to the TFIIE-TFIIH interaction in eukaryotes29. In Sulfolobus, TFE itself is composed of two subunits, further resembling eukaryotic TFIIE29,30. However, a key distinction is that archaeal transcription initiation does not require ATP hydrolysis, indicating that XPB’s translocase activity is dispensable in this context. Even if XPB does play a role in transcription in Archaea, it is not through its ATPase/translocase function31. These findings suggest that prior to the evolutionary divergence of Archaea and Eukaryotes, transcription initiation by RNA polymerase (structurally similar to RNA Pol II) could occur without the assistance of an ATP-dependent translocase. This has important evolutionary implications, particularly considering that although XPB is essential for transcription initiation at most human promoters, classical studies of the RNA Pol II transcription machinery and recent data indicate that a subset of promoters can initiate transcription without XPB activity, as discussed in the following section.

RNA polymerase II promoters that do not require XPB for transcription initiation

Since the pioneering in vitro studies of RNA Pol II transcription, it has been clear that DNA topology plays a critical role in transcription initiation. These early experiments, which reconstituted the basal transcription machinery using purified components, examined transcription from relatively strong promoters on both linear and supercoiled DNA templates. Notably, Parvin and Sharp, using the immunoglobulin heavy chain (IgH) promoter, demonstrated in vitro that transcription from supercoiled templates required only TBP, TFIIB, and RNA Pol II. In contrast, when using linear DNA templates with the IgH or Major Late Promoter (MLP), efficient transcription additionally required further general transcription factors, including TFIIH32. They also found that transcription efficiency increased with the degree of DNA supercoiling, suggesting that the free energy in supercoiled DNA was sufficient to drive open complex formation in the absence of TFIIH32. Similarly, Goodrich and Tjian observed that both linear and supercoiled templates supported transcription with RNA Pol II, TBP, TFIIB, and TFIIF. However, they found that TFIIE, TFIIH, and ATP hydrolysis were essential for promoter clearance on linear DNA, but not on supercoiled DNA33. These results from in vitro reconstituted systems suggested that, depending on the promoter, the translocase activity of XPB is not strictly required for DNA opening under conditions that favour spontaneous promoter melting.

Structural studies of the PIC have revealed that XPB, positioned ~20-30 bp downstream of the TSS, uses its ATP-dependent translocase activity to pull downstream DNA toward the PIC, thereby promoting strand separation at the TSS34. While XPB is essential for DNA opening at most human promoters, recent in vivo evidence suggests that a subset of promoters can initiate transcription independently of XPB activity (see below).

Triptolide (TPL) has been widely used as an inhibitor of XPB ATPase activity and, consequently, its translocase function. Although the possibility of secondary targets cannot be formally excluded, there is currently no compelling evidence for significant off-target effects. Notably, an XPB C342T mutant-substituting the cysteine residue to which TPL binds covalently-has been reported to confer resistance to TPL treatment2,35. Consistent with this, TPL treatment phenocopies mutations in p52 and XPB in Drosophila, supporting its specificity for the TFIIH-XPB axis36.

Genome-wide nascent transcription assays, including PRO-seq and GRO-seq, have shown that inhibition of XPB ATPase/translocase activity by TPL results in widespread transcriptional repression in mammalian cell lines37–39. However, a subset of promoters remains transcriptionally active even in the presence of TPL, indicating that XPB is not universally required for transcription initiation (see below).

In Saccharomyces cerevisiae, a conditional mutant in the Ssl2 gene, which encodes the XPB ortholog, led to reduced transcription in the majority of genes40. Interestingly, approximately 18% of genes remained unaffected. Analysis of their promoters revealed no significant differences in GC content, but when free energy (ΔG) was calculated across the region from +20 to +30 relative to the TATA box, these XPB-independent promoters exhibited less negative ΔG values, indicating a more unstable DNA duplex40. In fact, this study demonstrated that introducing mutations into the stable GAT1 promoter that mimic the unstable HIS4 promoter renders it functionally similar, eliminating its dependence on SSL240. This instability may facilitate DNA opening, enabling transcription initiation without the need for XPB activity. It is also noteworthy that promoters recognized by RNA polymerases I and III, which do not require ATP for transcription initiation, are typically characterized by intrinsic thermodynamic instability.

In addition to TPL, studies examining the role of TFIIH in transcription have used spironolactone (SP), an aldosterone antagonist that reduces nucleotide excision repair (NER) and transcription by selectively promoting degradation of the XPB subunit of TFIIH41. Notably, analyses in human fibroblasts in which XPB was either inhibited by TPL or depleted via SP-induced degradation, with nascent RNA synthesis measured by 5-ethynyl uridine (5EU) incorporation, showed that TPL nearly abolished global transcription, whereas residual transcription persisted in the absence of XPB. In this context, it has been suggested that TPL may exert a toxic effect on transcription initiation. Notably, in the complete absence of XPB, some genes can still be expressed. These observations indicate that a subset of promoters is capable of initiating transcription independently of XPB translocase activity. It is important to note that global transcriptional outcomes differ when SP induces XPB degradation compared with inhibition of XPB translocase activity by TPL. In this context, a genome-wide analysis using PRO-seq or GRO-seq in SP-treated cells would provide a comprehensive view of the direct effects on transcription initiation by SP.

Recent PRO-seq experiments performed in K562 cells, a broadly used human cell line derived from a patient with chronic myelogenous leukemia, treated with TPL have further confirmed that, although the majority of RNA Pol II-dependent transcripts are downregulated, a subset of genes is resistant to TPL, and some even show increased transcription42. Promoters of these TPL-insensitive genes were enriched for lower GC content, suggesting a correlation between DNA sequence composition and XPB independence. These findings collectively indicate that while XPB is crucial for the majority of transcription initiation events, a distinct class of XPB-independent promoters exists. Interestingly, both XPB-sensitive and XPB-resistant promoters to TPL exhibit a distinctive ΔG profile across their minimal promoter regions. Specifically, the region approximately −25 to −30 bp upstream of the TSS is relatively unstable, followed by a more stable segment between −20 and −5 bp. As expected, the TSS itself shows a less negative ΔG value across all promoter types, followed by a sharp drop immediately downstream (Fig. 3a). Around +10 bp downstream of the TSS, DNA instability increases markedly, peaks near +15 bp, and then rapidly stabilizes beyond this point.

Fig. 3. Thermodynamic profiles of stable and unstable promoters in human cells, and a meta-analysis of XPB and RNA polymerase II occupancy in cells treated or untreated with the XPB ATPase inhibitor triptolide (TPL).

Fig. 3

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a DNA free energy profiles at promoters that are independent of XPB translocase activity (energetically unstable) versus those that require XPB translocase activity. The ΔG data for stable and unstable promoters in human cells and in yeast were based on ref. 40,42. b Features of XPB-dependent and XPB-independent promoters. c Distribution of XPB around the TSS. At promoters that require XPB translocase activity, TPL treatment leads to increased XPB accumulation near the TSS, reflected by an enlarged peak. In contrast, at promoters that do not require XPB translocase activity for transcription initiation, XPB occupancy remains unaffected by TPL. RNA Pol II occupancy is also altered by TPL treatment. Under normal conditions, the major peak of RNA Pol II corresponds to the paused polymerase, mapping between +20 and +100 bp downstream of the TSS, while a smaller peak overlaps the TSS and represents initiating polymerase. Following TPL treatment, these peaks are inverted: the initiation peak becomes dominant, consistent with RNA Pol II stalling at the TSS.

This thermodynamic pattern suggests that the minimal RNA Pol II promoter encodes an intrinsic energy landscape that aligns with distinct functional subregions. Notably, the most unstable promoters (characterized by lower DNA duplex stability) can be transcribed efficiently without the need for XPB’s ATPase/translocase activity. This behavior resembles that of archaeal promoters and those transcribed by RNA polymerases I and III (Fig. 3a)40,42,43.

This promoter stability profile from +30 to -20 bp in human promoters appears to be largely independent of specific core promoter motifs, such as the TATA box or Downstream Promoter Element (DPE-like). However, promoters containing CpG islands (generally more GC-rich and thermodynamically stable) tend to require XPB translocase activity for transcription initiation42. Consistent with this, XPB ChIP-nexus44 and ChIP-exo42 experiments in Drosophila and human cells treated with TPL show that both XPB and RNA Pol II accumulate at the TSS in unstable promoters, but not in stable ones. This indicates that inhibition of XPB’s translocase activity does not affect the PIC dynamics at unstable promoters during transcription initiation (Fig. 3b).

What happens to the rest of TFIIH in the PIC when XPB activity is not required?

TFIIH plays multiple roles within the PIC beyond its well-known function in DNA unwinding through XPB. Notably, the CAK subcomplex (composed of CDK7, cyclin H, and MAT1) phosphorylates Ser5 of the heptapeptide repeats in the CTD of RNA Pol II. This Ser5 phosphorylation is a key signal for transcription initiation and is essential for recruiting RNA processing and chromatin-modifying complexes, as well as elongation and termination factors45,46.

In addition, TFIIH establishes a network of interactions with various components of the transcriptional machinery and with the Mediator complex during PIC assembly and activation. For example, the MAT1 and p62 subunits of TFIIH interact with the Rpb4 and Rpb7 subunits of RNA Pol II, while p62 also interfaces with TFIIE, an important player in DNA bubble formation at the TSS. MAT1 helps position CDK7 and cyclin H so that CAK activity can be stabilized by Mediator, facilitating CTD phosphorylation14–16.

Structurally, XPB interacts with XPD, MAT1, p52, and p8, while p52 contacts p34 and p44. Notably, p52 and p8 also modulate the ATPase activity of XPB (Fig. 1b and c)47,48. The absence of XPB, or mutations that disrupt its function, destabilizes this submodule of the complex, underscoring XPB’s architectural importance within TFIIH49–51. TFIIH also forms contacts with transcriptional regulators1, and several of its subunits (including XPB, p52, p44, and p34) interact with the +1 nucleosome, a key regulatory element located immediately downstream of the TSS. These interactions may play a role in facilitating nucleosome repositioning during transcription initiation21. Although XPB is essential and mutations in its ATPase domain are associated with human diseases such as xeroderma pigmentosum (XP) and trichothiodystrophy (TTD)52, it is now evident that not all promoters require XPB’s translocase activity for transcription initiation. However, this does not imply that XPB or TFIIH are dispensable in these contexts; rather, it reflects alternative pathways for achieving promoter opening at specific genes.

Indeed, structural and functional studies suggest that TFIIH must still be fully assembled and recruited to promoters where XPB activity is not required. XPB contributes to the three-dimensional architecture of the PIC, mediates protein-protein interactions essential for PIC stability, and anchors other TFIIH subunits in place. Moreover, the CAK subcomplex continues to perform its critical role in CTD phosphorylation, even when DNA opening does not depend on XPB. As mentioned before, in yeast, a conditional Ssl2 mutant retains transcription of ~18% of genes40. Because Ssl2 depletion is incomplete, residual Ssl2 may remain incorporated into TFIIH and the PIC, permitting transcription at a subset of promoters-particularly those with less stable architectures-similar to the effects observed with TPL. However, caution is warranted when interpreting results from pharmacological inhibition of transcription, including treatments with TPL or spironolactone, as off-target effects are often unavoidable. The development of novel XPB inhibitors will be valuable for enabling more robust and comparative analyses.

In summary, although the translocase activity of XPB may be bypassed at a subset of promoters, the complete TFIIH complex remains essential for proper PIC function and transcription initiation. XPB’s structural role and TFIIH’s multifaceted interactions with RNA Pol II, Mediator, and chromatin components ensure that TFIIH continues to play a central role in transcription regulation even when its catalytic activity is not required.

Concluding remarks and perspectives

In eukaryotic cells, XPB is essential for survival, not only because of its role in transcription initiation but also because of its involvement in DNA repair and additional functions of TFIIH, such as telomere maintenance and mitotic chromosome segregation. The discovery that a subset of promoters in both yeast and human cells can initiate transcription without the translocase activity of XPB raises important questions about the diversity and regulation of RNA Pol II transcription mechanisms.

As mentioned before, XPB mutations associated with human syndromes primarily impair transcription while still allowing organismal development52. Mutations in the translocase/ATPase domain affect both transcription and NER, are generally hypomorphic, and cause TTD and XP. In contrast, mutations in the DRD selectively disrupt NER and are linked to XP. Alleles mapping to the RED or ThM partially impair both transcription and NER and are associated with TTD and XP, whereas mutations in the C-terminal domain mainly affect transcription and result in TTD52. Therefore, an important question is whether these mutations preferentially affect genes with stable promoters, have a reduced impact on unstable promoters, and how this differential sensitivity relates to disease severity.

Based on what we know from archaeal transcription systems, the ancestral transcription initiation mechanism did not require ATP-dependent promoter melting by XPB. Thus, XPB’s translocase function to “open” the DNA around the TSS may have emerged during evolution to enhance the transcriptional adaptability of eukaryotes, enabling efficient initiation from promoters with higher DNA duplex stability or complex regulatory architectures. This evolutionary adaptation could have facilitated the fine-tuning of gene expression across varying environmental conditions and developmental stages.

To test this hypothesis, it would be valuable to experimentally manipulate promoter stability in a genomic context. For example, exchanging DNA motifs between stable (XPB-dependent) and unstable (XPB-independent) promoters directly in the genome of animal cells could reveal how intrinsic DNA properties influence transcription efficiency and regulation. While similar studies have been conducted in vitro using yeast promoters40, they did not preserve the native chromatin and genomic context of the modified promoters.

Interestingly, in XPB-independent promoters, particularly in yeast, no clear functional clustering has been observed, with the exception of genes encoding ribosomal proteins, which tend to be associated with unstable promoters40. In other cases, no obvious link to gene function has emerged. It is likely that genomic positioning, chromatin structure, and regulatory complexity contribute to the ability of some promoters to bypass XPB.

A further open question concerns the relationship between promoter stability and transcriptional dynamics, particularly transcriptional bursting53,54. Genome-wide studies have quantified the frequency and amplitude of transcription bursts, and it would be worth investigating whether these parameters differ between stable and unstable promoters. For example, XPB-independent promoters, being less stable, may sustain an open DNA conformation longer, potentially allowing faster or more frequent transcription reinitiation than stable promoters that rely on XPB-mediated DNA opening.

Addressing this will require integrative approaches combining chromatin context, promoter architecture, and real-time transcription dynamics to fully understand and elucidate the diversity of transcription initiation mechanisms in eukaryotes.

Methods: protein models

Protein structure predictions were performed using AlphaFold 3 via its public web server (https://alphafoldserver.com). All predictions used default server parameters. Five models were generated per job, and the top-ranked model (model_0) was used for all figures. Human XPB (UniProt P19447) was predicted as a monomer (server version 1; model seed: 263066388; pTM = 0.78). The XPB-p52-p8 submodule was predicted as a heterotrimer with ATP (P19447, Q92759, Q6ZYL4; server version 3; model seed: 996715364; pTM = 0.74; ipTM = 0.74). The human holo-TFIIH complex was predicted as a ten-subunit assembly comprising XPB (P19447), XPD (P18074), p62 (P32780), p44 (Q13888), p52 (Q92759), p34 (Q13889), p8 (Q6ZYL4), CDK7 (P50613), Cyclin H (P51946), and MAT1 (P51948) (server version 1; model seed: 1030715723; pTM = 0.66; ipTM = 0.63). Archaeal protein structures from Archaeoglobus fulgidus were predicted individually as monomers (server version 3): AfXPB (O29889; model seed: 1989036832; pTM = 0.80), AfTFB (O28970; model seed: 68890891; pTM = 0.76), and AfTBP (O29874; model seed: 1405468062; pTM = 0.91). AfTFE (O29501) was predicted as a homodimer (model seed: 165247829; pTM = 0.48; ipTM = 0.17). The experimental structure PDB: 8BVW (human PIC-Mediator complex with +1 nucleosome) was used and shown from two different orientations. Protein structures were visualized with UCSF ChimeraX55, and domain schematics were generated with IBS 2.056. All AlphaFold 3 models generated in this study are available upon request to the corresponding author (mario.zurita@ibt.unam.mx).

Figures and illustrations were created with BioRender (Created in BioRender. Calvario, E. (2025). https://BioRender.com/3108y6j).

Supplementary information

Transparent Peer Review file (661.6KB, pdf)

Acknowledgements

We thank Andrea Ortega for assistance. This work was funded by PAPIIT/UNAM project No. IN200124 to M.Z.

Author contributions

MZ: Conceived the study and wrote the manuscript. SCR: Contributed ideas and edited the manuscript. EC: Generated structural models using AlphaFold, edited the manuscript, and designed the figures.

Peer review

Peer review information

Communications Biology thanks Li Fan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Laura Rodríguez Pérez. A peer review file is available.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-10203-1.

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