The Long Road to Understanding RNAPII Transcription Initiation and Related Syndromes.

In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release.

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

Three-step mechanism of promoter escape by RNA polymerase II.

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.

Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.

When the +1 nucleosome gets in the way, TFIIH fails but does not fall.

In this issue of Molecular Cell, Abril-Garrido et al.1 used cryo-EM to uncover that the +1 nucleosome inhibits transcription by interfering with the function of the TFIIH translocase via mechanisms that depend on its position relative to the transcription start site.

Structural basis of transcription reduction by a promoter-proximal +1 nucleosome.

At active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal +1 nucleosome can reduce RNA synthesis in vivo and in vitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the +1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10 bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation.

Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs.

Erk1/2 activation contributes to mouse ES cell pluripotency. We found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation.

Lesion recognition by XPC, TFIIH and XPA in DNA excision repair.

Nucleotide excision repair removes DNA lesions caused by ultraviolet light, cisplatin-like compounds and bulky adducts1. After initial recognition by XPC in global genome repair or a stalled RNA polymerase in transcription-coupled repair, damaged DNA is transferred to the seven-subunit TFIIH core complex (Core7) for verification and dual incisions by the XPF and XPG nucleases2. Structures capturing lesion recognition by the yeast XPC homologue Rad4 and TFIIH in transcription initiation or DNA repair have been separately reported3-7. How two different lesion recognition pathways converge and how the XPB and XPD helicases of Core7 move the DNA lesion for verification are unclear. Here we report on structures revealing DNA lesion recognition by human XPC and DNA lesion hand-off from XPC to Core7 and XPA. XPA, which binds between XPB and XPD, kinks the DNA duplex and shifts XPC and the DNA lesion by nearly a helical turn relative to Core7. The DNA lesion is thus positioned outside of Core7, as would occur with RNA polymerase. XPB and XPD, which track the lesion-containing strand but translocate DNA in opposite directions, push and pull the lesion-containing strand into XPD for verification.

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes.

RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

Global regulation of promoter melting in naive lymphocytes.

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Does TFIIH move nucleosomes?

Transcription factor (TF) IIH is a factor involved in transcription, DNA repair, mitosis, and telomere stability. These functions stem from its helicase/ATPase and kinase activities. Recent reports on the structure and function of the transcription machinery, as well as chromosome compaction during mitosis, suggest that TFIIH also influences nucleosome movement, are explored here.

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus Pol II, which is 99.9% identical to human Pol II. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8 Å resolution, and resolve the structure of TFIIH at 2.9-4.0 Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Structural basis of human transcription-DNA repair coupling.

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

Promoter Distortion and Opening in the RNA Polymerase II Cleft.

Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening.

Interplay of the Tfb1 pleckstrin homology domain with Rad2 and Rad4 in transcription coupled and global genomic nucleotide excision repair.

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PHD-binding segments (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function within one module for damage recognition in TCR and GGR. Rad2 PB1 protects Tfb1 from degradation and interacts with Tfb1 PHD at a basal level, presumably within transcription preinitiation complexes when NER is inactive. During a late step of NER, the interaction between Rad2 PB1 and Tfb1 PHD augments, enabling efficient TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby weakening the protection of Tfb1 from degradation and enabling rapid augmentation of their interactions within TCR and GGR complexes. Our results shed new light on NER mechanisms.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

TFIIH mutations can impact on translational fidelity of the ribosome.

TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.

Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.