RESUMO
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.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Fosforilação , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografia por Raios X , RNA Polimerase II/metabolismo , RNA Polimerase II/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Domínios Proteicos , Proteínas de Ciclo CelularRESUMO
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.
Assuntos
Fosfoproteínas , Regiões Promotoras Genéticas , RNA Polimerase II , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Fatores de Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIB/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIA/genética , Transcrição Gênica , Elongação da Transcrição Genética , RNA/metabolismo , RNA/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genéticaRESUMO
The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.
Assuntos
Domínios de Homologia à Plecstrina , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , Estrutura Terciária de Proteína , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Drug repositioning is one of the most effective approaches towards drug discovery and development. It involves the identification of new therapeutic indications of existing drugs. The present study evaluated several drugs for their ability to modulate activity of the p8 subunit of TFIIH complex. Negative modulation of p8 subunit activity disrupts protein-protein interactions (PPIs) among the subunits of TFIIH complex, and thereby the TFIIH-associated functions. TFIIH complex has key role in the transcription and nucleotide excision repair activity in cancerous cells. TFIIH complex has emerged as a privileged drug target in anticancer research. Out of 60 drugs, amlopipine (13), diltiazem (16), gemfibrozil (19), levocitrizine dihydrochloride (20), losartan potassium (22), clorthalidone (24), and escitalopram (28) showed interactions with subunit p8 in the ligand-protein binding and chemical shift perturbation studies. The Kd values were found to be between 0.25 and 1 mM. These drugs also caused thermal destabilization of the subunit p8 by negatively shifting the melting temperature by ≥ 2 °C. Molecular docking studies indicated the interaction of these drugs with important residues of p8-p52 complex, such as Glu48, Lys51, Glu496, and Glu455 via non-covalent interactions. This study has thereby identified 7 drugs that can be investigated further as potential anticancer drugs.
Assuntos
Antineoplásicos , Reposicionamento de Medicamentos , Antineoplásicos/farmacologia , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62-RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62-RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.
Assuntos
RNA Polimerase II/química , Fator de Transcrição TFIIH/química , Sítios de Ligação , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Domínios de Homologia à Plecstrina , Ligação Proteica , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismoRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Gênica , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , UbiquitinaçãoRESUMO
The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.
Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genéticaRESUMO
The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.
Assuntos
Antineoplásicos/química , Bibliotecas de Moléculas Pequenas/química , Fator de Transcrição TFIIH/antagonistas & inibidores , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidade , Eletricidade Estática , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismoRESUMO
The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.
Assuntos
Complexo Mediador/química , RNA Polimerase II/química , Fator de Transcrição TFIID/química , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Humanos , Complexo Mediador/metabolismo , Subunidade 1 do Complexo Mediador/química , Modelos Moleculares , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , RNA Polimerase II/metabolismo , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
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.
Assuntos
DNA/química , DNA/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Animais , Pareamento de Bases , DNA/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Mamíferos/genética , Modelos Moleculares , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , TATA Box/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição GenéticaRESUMO
Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
Assuntos
Complexo Mediador/química , RNA Polimerase II/química , Fatores Genéricos de Transcrição/química , Iniciação da Transcrição Genética , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
TFIIH is a crucial transcription and DNA repair factor consisting of the seven-subunit core. The core subunit p62 contains a pleckstrin homology domain (PH-D), which is essential for locating TFIIH at transcription initiation and DNA damage sites, and two BSD (BTF2-like transcription factors, synapse-associated proteins and DOS2-like proteins) domains. A recent cryo-electron microscopy (cryo-EM) structure of human TFIIH visualized most parts of core, except for the PH-D. Here, by nuclear magnetic resonance spectroscopy we have established the solution structure of human p62 PH-D connected to the BSD1 domain by a highly flexible linker, suggesting the flexibility of PH-D in TFIIH. Based on this dynamic character, the PH-D was modeled in the cryo-EM structure to obtain the whole human TFIIH core structure, which indicates that the PH-D moves around the surface of core with a specific but limited spatial distribution; these dynamic structures were refined by molecular dynamics (MD) simulations. Furthermore, we built models, also refined by MD simulations, of TFIIH in complex with five p62-binding partners, including transcription factors TFIIEα, p53 and DP1, and nucleotide excision repair factors XPC and UVSSA. The models explain why the PH-D is crucially targeted by these factors, which use their intrinsically disordered acidic regions for TFIIH recruitment.
Assuntos
Fator de Transcrição TFIIH/química , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Domínios de Homologia à Plecstrina , Domínios ProteicosRESUMO
The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.
Assuntos
Adenosina Trifosfatases/química , Chaetomium/química , DNA/genética , Proteínas Fúngicas/química , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.
Assuntos
Dano ao DNA , Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição Genética , DNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Fator de Transcrição TFIIH/química , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds-ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3'-overhang while Bax1 interacts with the 5'-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.
Assuntos
DNA Helicases/química , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Desoxirribonucleases/química , Pareamento Incorreto de Bases , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Sulfolobaceae/enzimologia , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismoRESUMO
XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.
Assuntos
Dano ao DNA , DNA/química , Modelos Moleculares , Proteína de Xeroderma Pigmentoso Grupo A/química , Aminoácidos , Sítios de Ligação , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Modelos Biológicos , Conformação Molecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Eukaryotic transcription factor IIH (TFIIH) is a 500 kDa-multiprotein complex that harbors two SF2-family DNA-dependent ATPase/helicase subunits and the kinase activity of Cyclin-dependent kinase 7. TFIIH serves as a general transcription factor for transcription initiation by eukaryotic RNA polymerase II and plays an important role in nucleotide excision DNA repair. Aiming to understand the molecular mechanisms of its function and regulation in two key cellular pathways, the high-resolution structure of TFIIH has been pursued for decades. Recent breakthroughs, largely enabled by methodological advances in cryo-electron microscopy, have finally revealed the structure of TFIIH and its interactions in the context of the Pol II-pre-initiation complex, and provide a first glimpse of a TFIIH-containing assembly in DNA repair. Here, we review and discuss these recent structural insights and their functional implications.
Assuntos
Microscopia Crioeletrônica , Relação Quantitativa Estrutura-Atividade , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , DNA/química , DNA/metabolismo , Reparo do DNA , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transcrição Gênica , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly's global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE-p62 interactions that link core-PIC to TFIIH. p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations associated with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.
Assuntos
Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Iniciação da Transcrição Genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Modelos Moleculares , Mapas de Interação de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fator de Transcrição TFIIH/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/químicaRESUMO
Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 Å resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.
Assuntos
DNA Helicases/química , Proteínas de Ligação a DNA/química , Subunidade p52 de NF-kappa B/química , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/fisiologia , Proteínas de Ciclo Celular/química , Microscopia Crioeletrônica , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Células HeLa , Humanos , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Fator de Transcrição TFIIH/genética , Fatores de Transcrição/química , Transcrição Gênica , Proteína Grupo D do Xeroderma Pigmentoso/químicaRESUMO
The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.