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1.
Cell ; 175(5): 1393-1404.e11, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454648

RESUMO

Ribonuclease (RNase) P is a ubiquitous ribozyme that cleaves the 5' leader from precursor tRNAs. Here, we report cryo-electron microscopy structures of the human nuclear RNase P alone and in complex with tRNAVal. Human RNase P is a large ribonucleoprotein complex that contains 10 protein components and one catalytic RNA. The protein components form an interlocked clamp that stabilizes the RNA in a conformation optimal for substrate binding. Human RNase P recognizes the tRNA using a double-anchor mechanism through both protein-RNA and RNA-RNA interactions. Structural comparison of the apo and tRNA-bound human RNase P reveals that binding of tRNA induces a local conformational change in the catalytic center, transforming the ribozyme into an active state. Our results also provide an evolutionary model depicting how auxiliary RNA elements in bacterial RNase P, essential for substrate binding, and catalysis, were replaced by the much more complex and multifunctional protein components in higher organisms.


Assuntos
Microscopia Crioeletrônica , RNA de Transferência/química , Ribonuclease P/química , Sítios de Ligação , Evolução Molecular , Células HEK293 , Holoenzimas/química , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Terciária de Proteína , RNA de Transferência/metabolismo , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismo
2.
Molecules ; 28(13)2023 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-37446716

RESUMO

Sensitive detection of nitrogen dioxide (NO2) is of significance in many areas for health and environmental protections. In this work, we developed an efficient NO2 sensor that can respond within seconds at room temperature, and the limit of detection (LOD) is as low as 100 ppb. Coating cyano-substituted poly(p-phenylene vinylene) (CN-PPV) films on graphene (G) layers can dope G sheets effectively to a heavy n state. The influences of solution concentrations and annealing temperatures on the n-doping effect were investigated in detail. The CN-PPV-G transistors fabricated with the optimized parameters demonstrate active sensing abilities toward NO2. The n-doping state of CN-PPV-G is reduced dramatically by NO2, which is a strong p-doping compound. Upon exposure to 25 ppm of NO2, our CN-PPV-G sensors react in 10 s, indicating it is almost an immediate response. LOD is determined as low as 100 ppb. The ultrahigh responding speed and low LOD are not affected in dry air. Furthermore, cycling use of our sensors can be realized through simple annealing. The superior features shown by our CN-PPV-G sensors are highly desired in the applications of monitoring the level of NO2 in situ and setting immediate alarms. Our results also suggest that transfer curves of transistors can react very promptly to the stimulus of target gas and, thus, are very promising in the development of fast-response sensing devices although the response values may not reach maximum as a tradeoff.


Assuntos
Grafite , Dióxido de Nitrogênio , Limite de Detecção , Temperatura
3.
RNA Biol ; 19(1): 246-255, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35133940

RESUMO

RNA polymerase III (Pol III) is a large multisubunit complex conserved in all eukaryotes that plays an essential role in producing a variety of short non-coding RNAs, such as tRNA, 5S rRNA and U6 snRNA transcripts. Pol III comprises of 17 subunits in both yeast and human with a 10-subunit core and seven peripheral subunits. Because of its size and complexity, Pol III has posed a formidable challenge to structural biologists. The first atomic cryogenic electron microscopy structure of yeast Pol III leading to the canonical view was reported in 2015. Within the last few years, the optimization of endogenous extract and purification procedure and the technical and methodological advances in cryogenic electron microscopy, together allow us to have a first look at the unprecedented details of human Pol III organization. Here, we look back on the structural studies of human Pol III and discuss them in the light of our current understanding of its role in eukaryotic transcription.


Assuntos
Modelos Moleculares , Conformação Proteica , RNA Polimerase III/química , RNA Polimerase III/metabolismo , Archaea/enzimologia , Sequência Conservada , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , RNA Polimerase III/genética , Relação Estrutura-Atividade , Leveduras/enzimologia
4.
Biochim Biophys Acta Bioenerg ; 1858(4): 325-335, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28188780

RESUMO

The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αß)3 trimer and (αß)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and ß subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.


Assuntos
Cianobactérias/química , Fotossíntese , Ficobiliproteínas/química , Ficocianina/química , Proteína C/química , Temperatura Baixa , Cristalização , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estabilidade Proteica
5.
Proc Natl Acad Sci U S A ; 111(31): 11347-52, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25059720

RESUMO

p150(glued) belongs to a group of proteins accumulating at microtubule plus ends (+TIPs). It plays a key role in initiating retrograde transport by recruiting and tethering endosomes and dynein to microtubules. p150(glued) contains an N-terminal microtubule-binding cytoskeleton-associated protein glycine-rich (CAP-Gly) domain that accelerates tubulin polymerization. Although this copolymerization is well-studied using light microscopic techniques, structural consequences of this interaction are elusive. Here, using electron-microscopic and spectroscopic approaches, we provide a detailed structural view of p150(glued) CAP-Gly binding to microtubules and tubulin. Cryo-EM 3D reconstructions of p150(glued)-CAP-Gly complexed with microtubules revealed the recognition of the microtubule surface, including tubulin C-terminal tails by CAP-Gly. These binding surfaces differ from other retrograde initiation proteins like EB1 or dynein, which could facilitate the simultaneous attachment of all accessory components. Furthermore, the CAP-Gly domain, with its basic extensions, facilitates lateral and longitudinal interactions of tubulin molecules by covering the tubulin acidic tails. This shielding effect of CAP-Gly and its basic extensions may provide a molecular basis of the roles of p150(glued) in microtubule dynamics.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Temperatura Baixa , Microscopia Crioeletrônica , Complexo Dinactina , Dineínas/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Processamento de Imagem Assistida por Computador , Cinética , Microtúbulos/ultraestrutura , Peptídeos/química , Peptídeos/metabolismo , Polimerização , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-Atividade , Tubulina (Proteína)/ultraestrutura
6.
Nat Commun ; 15(1): 4622, 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38816438

RESUMO

The 5'-end capping of nascent pre-mRNA represents the initial step in RNA processing, with evidence demonstrating that guanosine addition and 2'-O-ribose methylation occur in tandem with early steps of transcription by RNA polymerase II, especially at the pausing stage. Here, we determine the cryo-EM structures of the paused elongation complex in complex with RNGTT, as well as the paused elongation complex in complex with RNGTT and CMTR1. Our findings show the simultaneous presence of RNGTT and the NELF complex bound to RNA polymerase II. The NELF complex exhibits two conformations, one of which shows a notable rearrangement of NELF-A/D compared to that of the paused elongation complex. Moreover, CMTR1 aligns adjacent to RNGTT on the RNA polymerase II stalk. Our structures indicate that RNGTT and CMTR1 directly bind the paused elongation complex, illuminating the mechanism by which 5'-end capping of pre-mRNA during transcriptional pausing.


Assuntos
Microscopia Crioeletrônica , Capuzes de RNA , RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/química , Capuzes de RNA/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Humanos , Ligação Proteica , Modelos Moleculares , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
7.
Nat Commun ; 15(1): 7092, 2024 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-39154037

RESUMO

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.


Assuntos
Microscopia Crioeletrônica , Lisina Acetiltransferase 5 , Humanos , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/química , Lisina Acetiltransferase 5/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , DNA Helicases/metabolismo , DNA Helicases/química , Modelos Moleculares , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Ligação Proteica , Multimerização Proteica , Proteínas que Contêm Bromodomínio , Proteínas Adaptadoras de Transdução de Sinal
8.
Cell Discov ; 10(1): 15, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331872

RESUMO

Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A-H2B dimer with H2A.Z-H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C. The motor module adopts distinct nucleosome binding modes in the apo (nucleotide-free), ADP-bound, and ADP-BeFx-bound states, suggesting that ATPase-driven movement destabilizes H2A-H2B by unwrapping the entry DNA and pulls H2A-H2B out of nucleosome through the ZNHIT1 subunit. Structure-guided chromatin immunoprecipitation sequencing analysis confirmed the requirement of H2A-contacting ZNHIT1 in maintaining H2A.Z occupancy on the genome. Our study provides structural insights into the mechanism of H2A-H2A.Z exchange mediated by SRCAP-C.

9.
Science ; 382(6677): eadi5120, 2023 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-38127763

RESUMO

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.


Assuntos
RNA , Fatores Genéricos de Transcrição , Iniciação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , RNA/biossíntese , RNA Polimerase II/química , Fatores Genéricos de Transcrição/metabolismo , Humanos , Animais , Sus scrofa , Microscopia Crioeletrônica , Filmes Cinematográficos
10.
Gene ; 821: 146282, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35149153

RESUMO

RNA polymerase (Pol) III is responsible for the transcription of tRNAs, 5S rRNA, U6 snRNA, and other non-coding RNAs. Transcription factors such as TFIIIA, -B, -C, SNAPc, and Maf1 are required for promoter recognition, promoter opening, and Pol III activity regulation. Recent developments in cryo-electron microscopy and advanced purification approaches for endogenous multi-subunit complexes accelerated structural studies resulting in detailed structural insights which allowed an in-depth understanding of the molecular mechanisms underlying Pol III transcription. Here, we summarize structural data on Pol III and its regulating factors providing a three-dimensional framework to guide further analysis of RNA polymerase III.


Assuntos
Complexos Multiproteicos/química , RNA Polimerase III/metabolismo , Fatores de Transcrição/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase III/química , Fatores de Transcrição/química
11.
Nat Commun ; 13(1): 7644, 2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36496390

RESUMO

BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite previous structural studies and recent advance of SWI/SNF family complexes, it remains incompletely understood how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions. PBAF-nucleosome structure reveals six histone-binding domains and four DNA-binding domains/modules, the majority of which directly bind histone/DNA. This multivalent nucleosome-binding pattern, not observed in previous studies, suggests that PBAF may integrate comprehensive chromatin information to target genomic loci for function. Our study reveals molecular organization of subunits and histone/DNA-binding domains/modules in PBAF-nucleosome complex and provides structural insights into PBAF-mediated nucleosome association complimentary to the recently reported PBAF-nucleosome structure.


Assuntos
Proteínas Cromossômicas não Histona , Nucleossomos , Animais , Humanos , Nucleossomos/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Histonas/genética , Histonas/metabolismo , Montagem e Desmontagem da Cromatina , Mamíferos/genética
12.
Nat Struct Mol Biol ; 28(2): 220-227, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33558766

RESUMO

RNA polymerase III (Pol III) synthesizes structured, essential small RNAs, such as transfer RNA, 5S ribosomal RNA and U6 small nuclear RNA. Pol III, the largest nuclear RNA polymerase, is composed of a conserved core region and eight constitutive regulatory subunits, but how these factors jointly regulate Pol III transcription remains unclear. Here, we present cryo-EM structures of human Pol III in both apo and elongating states, which unveil both an orchestrated movement during the apo-to-elongating transition and an unexpected apo state in which the RPC7 subunit tail occupies the DNA-RNA-binding cleft of Pol III, suggesting that RPC7 plays important roles in both autoinhibition and transcription initiation. The structures also reveal a proofreading mechanism for the TFIIS-like subunit RPC10, which stably retains its catalytic position in the secondary channel, explaining the high fidelity of Pol III transcription. Our work provides an integrated picture of the mechanism of Pol III transcription regulation.


Assuntos
Modelos Moleculares , RNA Polimerase III/química , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Conformação Proteica , RNA Polimerase III/ultraestrutura , Transcrição Gênica
13.
Nat Commun ; 10(1): 2617, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197137

RESUMO

Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5' maturation. Here we report the cryo-EM structures of Methanocaldococcus jannaschii (Mja) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of MjaRNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3'-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by MjaRNase P. The overall organization of MjaRNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.


Assuntos
Proteínas Arqueais/ultraestrutura , Ribonuclease P/ultraestrutura , Proteínas Arqueais/metabolismo , Biocatálise , Microscopia Crioeletrônica , Holoenzimas/ultraestrutura , Methanocaldococcus/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , Ribonuclease P/metabolismo
15.
Nat Commun ; 9(1): 4684, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30409972

RESUMO

The cilium is an organelle used for motility and cellular signaling. Intraflagellar transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. How IFT controls the movement of ciliary components is currently poorly understood. IFT172 is the largest IFT subunit essential for ciliogenesis. Due to its large size, the characterization of IFT172 has been challenging. Using giant unilamellar vesicles (GUVs), we show that IFT172 is a membrane-interacting protein with the ability to remodel large membranes into small vesicles. Purified IFT172 has an architecture of two globular domains with a long rod-like protrusion, resembling the domain organization of coatomer proteins such as COPI-II or clathrin. IFT172 adopts two different conformations that can be manipulated by lipids or detergents: 1) an extended elongated conformation and 2) a globular closed architecture. Interestingly, the association of IFT172 with membranes is mutually exclusive with IFT57, implicating multiple functions for IFT172 within IFT.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Flagelos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Membrana Celular/ultraestrutura , Chlamydomonas , Lipídeos/química , Lipossomos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo
16.
Sci Rep ; 7(1): 8542, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819147

RESUMO

Nitrogen is one of the most important nutrients needed for plants and algae to survive, and the photosynthetic ability of algae is related to nitrogen abundance. Red algae are unique photosynthetic eukaryotic organisms in the evolution of algae, as they contain phycobilisomes (PBSs) on their thylakoid membranes. In this report, the in vivo chlorophyll (Chl) a fluorescence kinetics of nitrogen-starved Porphyridium cruentum were analyzed to determine the effects of nitrogen deficiency on photosynthetic performance using a multi-color pulse amplitude modulation (PAM) chlorophyll fluorometer. Due to nitrogen starvation, the photochemical efficiency of PSII and the activity of PSII reaction centers (RCs) decreased, and photoinhibition of PSII occurred. The water-splitting system on the donor side of PSII was seriously impacted by nitrogen deficiency, leading to the inactivation of the oxygen-evolving complex (OEC) and decreased light energy conversion efficiency. In nitrogen-starved cells, a higher proportion of energy was used for photochemical reactions, and thermal dissipation was reduced, as shown by qP and qN. The ability of nitrogen-starved cells to tolerate and resist high photon flux densities was weakened. Our results showed that the photosynthetic performance of P. cruentum was severely impacted by nitrogen deficiency.


Assuntos
Proteínas de Algas/metabolismo , Clorofila A/metabolismo , Nitrogênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Porphyridium/metabolismo , Clorofila A/química , Fluorescência , Fluorometria/métodos , Luz , Oxigênio/metabolismo , Processos Fotoquímicos/efeitos da radiação , Fotossíntese/efeitos da radiação
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