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1.
Mol Cell ; 81(16): 3368-3385.e9, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34375583

RESUMO

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , RNA/genética , Fatores de Transcrição/genética , Adenosina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Elementos Reguladores de Transcrição/genética , Ativação Transcricional/genética
2.
Science ; 373(6555): 662-673, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353949

RESUMO

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias/genética , RNA Longo não Codificante/genética , Acetilgalactosamina , Animais , Biopterina/análogos & derivados , Biopterina/metabolismo , Biopterina/uso terapêutico , Dieta , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Fenilalanina/administração & dosagem , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêutico
3.
Cell Res ; 31(8): 861-885, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34108665

RESUMO

The molecular basis underlying the interaction between retrotransposable elements (RTEs) and the human genome remains poorly understood. Here, we profiled N6-methyladenosine (m6A) deposition on nascent RNAs in human cells by developing a new method MINT-Seq, which revealed that many classes of RTE RNAs, particularly intronic LINE-1s (L1s), are strongly methylated. These m6A-marked intronic L1s (MILs) are evolutionarily young, sense-oriented to hosting genes, and are bound by a dozen RNA binding proteins (RBPs) that are putative novel readers of m6A-modified RNAs, including a nuclear matrix protein SAFB. Notably, m6A positively controls the expression of both autonomous L1s and co-transcribed L1 relics, promoting L1 retrotransposition. We showed that MILs preferentially reside in long genes with critical roles in DNA damage repair and sometimes in L1 suppression per se, where they act as transcriptional "roadblocks" to impede the hosting gene expression, revealing a novel host-weakening strategy by the L1s. In counteraction, the host uses the SAFB reader complex to bind m6A-L1s to reduce their levels, and to safeguard hosting gene transcription. Remarkably, our analysis identified thousands of MILs in multiple human fetal tissues, enlisting them as a novel category of cell-type-specific regulatory elements that often compromise transcription of long genes and confer their vulnerability in neurodevelopmental disorders. We propose that this m6A-orchestrated L1-host interaction plays widespread roles in gene regulation, genome integrity, human development and diseases.

4.
Sci Adv ; 7(15)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827808

RESUMO

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

5.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649230

RESUMO

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.


Assuntos
Endorribonucleases , Splicing de RNA/genética , Estabilidade de RNA/genética , RNA Fúngico , RNA Mensageiro , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Sci Adv ; 7(3)2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33523904

RESUMO

The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic mutations causative for neurodevelopmental disorders and cancer congregate in CKM subunits. However, the structure of the intact CKM and the mechanism by which Cdk8 is non-canonically activated and functionally affected by oncogenic CKM alterations are poorly understood. Here, we report a cryo-electron microscopy structure of Saccharomyces cerevisiae CKM that redefines prior CKM structural models and explains the mechanism of Med12-dependent Cdk8 activation. Med12 interacts extensively with CycC and activates Cdk8 by stabilizing its activation (T-)loop through conserved Med12 residues recurrently mutated in human tumors. Unexpectedly, Med13 has a characteristic Argonaute-like bi-lobal architecture. These findings not only provide a structural basis for understanding CKM function and pathological dysfunction, but also further impute a previously unknown regulatory mechanism of Mediator in transcriptional modulation through its Med13 Argonaute-like features.

7.
Elife ; 72018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29460780

RESUMO

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the ß subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and ß2. Together, these structures support a model for RNR inhibition in which ß2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.


Assuntos
Multimerização Proteica , Ribonucleotídeo Redutases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Microscopia Crioeletrônica , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ribonucleotídeo Redutases/metabolismo
8.
Nature ; 544(7649): 196-201, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28241144

RESUMO

The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.


Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Complexo Mediador/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/ultraestrutura , Relação Estrutura-Atividade
9.
J Biol Chem ; 291(52): 26886-26898, 2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-27821593

RESUMO

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.


Assuntos
Holoenzimas/metabolismo , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/genética , Humanos , Complexo Mediador/química , Complexo Mediador/genética , Ligação Proteica , Conformação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição Genética
10.
Proc Natl Acad Sci U S A ; 112(44): 13543-8, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26483468

RESUMO

The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.


Assuntos
DNA/química , Complexos Multiproteicos/química , RNA Polimerase II/química , Fatores de Transcrição/química , Transcrição Genética , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TATA Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/ultraestrutura
11.
Nat Chem ; 6(12): 1065-71, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25411884

RESUMO

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here, we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Šdiameter, cube-shaped cage with large openings into a 130 Šdiameter inner cavity. A crystal structure of the cage showed atomic-level agreement with the designed model, while electron microscopy, native mass spectrometry and small angle X-ray scattering revealed alternative assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will probably require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.


Assuntos
Proteínas/química , Cristalografia por Raios X , Peso Molecular , Porosidade , Espalhamento de Radiação
12.
Nat Cell Biol ; 16(9): 852-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25173975

RESUMO

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Animais , Sistema Livre de Células , Escherichia coli , Humanos , Metáfase , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Xenopus laevis
13.
Cell ; 157(6): 1430-1444, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24882805

RESUMO

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.


Assuntos
Complexo Mediador/química , Complexo Mediador/ultraestrutura , Microscopia Crioeletrônica , Células HeLa , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
J Am Chem Soc ; 135(20): 7738-43, 2013 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-23621606

RESUMO

Designing protein molecules that self-assemble into complex architectures is an outstanding goal in the area of nanobiotechnology. One design strategy for doing this involves genetically fusing together two natural proteins, each of which is known to form a simple oligomer on its own (e.g., a dimer or trimer). If two such components can be fused in a geometrically predefined configuration, that designed subunit can, in principle, assemble into highly symmetric architectures. Initial experiments showed that a 12-subunit tetrahedral cage, 16 nm in diameter, could be constructed following such a procedure [Padilla, J. E.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2217; Lai, Y. T.; et al. Science 2012, 336, 1129]. Here we characterize multiple crystal structures of protein cages constructed in this way, including cages assembled from two mutant forms of the same basic protein subunit. The flexibilities of the designed assemblies and their deviations from the target model are described, along with implications for further design developments.


Assuntos
Nanoestruturas/química , Proteínas/química , Cristalografia por Raios X , Modelos Moleculares , Mutação , Tamanho da Partícula , Conformação Proteica , Proteínas/genética , Propriedades de Superfície
16.
Nat Struct Mol Biol ; 20(5): 611-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23563140

RESUMO

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.


Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Regulação Fúngica da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Quinase 8 Dependente de Ciclina/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Complexo Mediador/química , Microscopia Eletrônica , Ligação Proteica , RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/química
17.
Nature ; 475(7355): 240-3, 2011 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-21725323

RESUMO

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.


Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , Saccharomyces cerevisiae/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Fosforilação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo
18.
J Mol Biol ; 393(5): 1056-69, 2009 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-19744498

RESUMO

Helicase loading factors are thought to transfer the hexameric ring-shaped helicases onto the replication fork during DNA replication. However, the mechanism of helicase transfer onto DNA remains unclear. In Bacillus subtilis, the protein DnaI, which belongs to the AAA+ family of ATPases, is responsible for delivering the hexameric helicase DnaC onto DNA. Here we investigated the interaction between DnaC and DnaI from Geobacillus kaustophilus HTA426 (GkDnaC and GkDnaI, respectively) and determined that GkDnaI forms a stable complex with GkDnaC with an apparent stoichiometry of GkDnaC(6)-GkDnaI(6) in the absence of ATP. Surface plasmon resonance analysis indicated that GkDnaI facilitates loading of GkDnaC onto single-stranded DNA (ssDNA) and supports complex formation with ssDNA in the presence of ATP. Additionally, the GkDnaI C-terminal AAA+ domain alone could bind ssDNA, and binding was modulated by nucleotides. We also determined the crystal structure of the C-terminal AAA+ domain of GkDnaI in complex with ADP at 2.5 A resolution. The structure not only delineates the binding of ADP in the expected Walker A and B motifs but also reveals a positively charged region that may be involved in ssDNA binding. These findings provide insight into the mechanism of replicative helicase loading onto ssDNA.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , DNA Helicases/química , Replicação do DNA , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Cristalografia por Raios X , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície
19.
Nucleic Acids Res ; 37(3): 804-14, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19074952

RESUMO

DNA helicases are motor proteins that play essential roles in DNA replication, repair and recombination. In the replicative hexameric helicase, the fundamental reaction is the unwinding of duplex DNA; however, our understanding of this function remains vague due to insufficient structural information. Here, we report two crystal structures of the DnaB-family replicative helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in the apo-form and bound to single-stranded DNA (ssDNA). The GkDnaC-ssDNA complex structure reveals that three symmetrical basic grooves on the interior surface of the hexamer individually encircle ssDNA. The ssDNA-binding pockets in this structure are directed toward the N-terminal domain collar of the hexameric ring, thus orienting the ssDNA toward the DnaG primase to facilitate the synthesis of short RNA primers. These findings provide insight into the mechanism of ssDNA binding and provide a working model to establish a novel mechanism for DNA translocation at the replication fork.


Assuntos
Proteínas de Bactérias/química , DNA Helicases/química , DNA de Cadeia Simples/química , Modelos Moleculares , Bacillaceae/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , Estrutura Terciária de Proteína
20.
Nucleic Acids Res ; 35(20): 6984-94, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17940099

RESUMO

FOXO3a is a transcription factor of the FOXO family. The FOXO proteins participate in multiple signaling pathways, and their transcriptional activity is regulated by several post-translational mechanisms, including phosphorylation, acetylation and ubiquitination. Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics. To understand how FOXO mediate transcriptional activity, we report here the 2.7 A crystal structure of the DNA-binding domain of FOXO3a (FOXO3a-DBD) bound to a 13-bp DNA duplex containing a FOXO consensus binding sequence (GTAAACA). Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins. In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.


Assuntos
DNA/química , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/metabolismo , Proteína Forkhead Box O3 , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
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