Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.

Elf1 promotes transcription-coupled repair in yeast by using its C-terminal domain to bind TFIIH.

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.

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.

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.

Persistent TFIIH binding to non-excised DNA damage causes cell and developmental failure.

Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.

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.

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.

The Link of mRNA and rRNA Transcription by PUF60/FIR through TFIIH/P62 as a Novel Therapeutic Target for Cancer.

The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.