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1.
Nat Commun ; 12(1): 3883, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162887

RESUMO

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , DNA Fúngico/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070827

RESUMO

Precocious dissociation of sisters 5 (PDS5) is an associate protein of cohesin that is conserved from yeast to humans. It acts as a regulator of the cohesin complex and plays important roles in various cellular processes, such as sister chromatid cohesion, DNA damage repair, gene transcription, and DNA replication. Vertebrates have two paralogs of PDS5, PDS5A and PDS5B, which have redundant and unique roles in regulating cohesin functions. Herein, we discuss the molecular characteristics and functions of PDS5, as well as the effects of its mutations in the development of diseases and their relevance for novel therapeutic strategies.


Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Síndrome de Cornélia de Lange/metabolismo , Síndrome de Cornélia de Lange/patologia , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Troca de Cromátide Irmã , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

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/genética
4.
Nat Commun ; 12(1): 3483, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108481

RESUMO

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Adenosina Trifosfatases/química , Compostos de Boro/química , Proteínas de Saccharomyces cerevisiae/química , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Compostos de Boro/farmacologia , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Mutação , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 12(1): 2889, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001871

RESUMO

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Sítios de Ligação/genética , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Endocitose/genética , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
6.
Nat Commun ; 12(1): 2763, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980827

RESUMO

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/química , Exodesoxirribonucleases/química , Modelos Moleculares , Complexos Multiproteicos , Mutação , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
7.
Nat Commun ; 12(1): 2220, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850123

RESUMO

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Complexo Mediador/química , Complexo Mediador/genética , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
8.
Biochem Biophys Res Commun ; 555: 115-120, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33845395

RESUMO

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.


Assuntos
Cinesina/química , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Cinesina/genética , Mutação , Proteínas de Saccharomyces cerevisiae/genética
9.
Methods Mol Biol ; 2281: 151-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847957

RESUMO

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of select DNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-L-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.


Assuntos
Azidas/síntese química , DNA de Cadeia Simples/metabolismo , Fenilalanina/análogos & derivados , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Azidas/química , Replicação do DNA , Corantes Fluorescentes/química , Modelos Moleculares , Fenilalanina/síntese química , Fenilalanina/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos
10.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916983

RESUMO

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Algoritmos , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinética , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
11.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917059

RESUMO

Tubules of the endoplasmic reticulum (ER) spread into the buds of yeast by an actin-based mechanism and, upon entry, become attached to the polarisome, a proteinaceous micro-compartment below the tip of the bud. The minimal tether between polarisome and cortical ER is formed by a protein complex consisting of Epo1, a member of the polarisome, Scs2, a membrane protein of the ER and Cdc42 guanosine triphosphatase-activating protein Bem3. Here, we report the crystal structure of a complex between Epo1 and Bem3. In addition, we characterize through the hydrogen/deuterium (H/D) exchange assay the interface between Scs2 and Epo1. Our findings provide a first structural insight into the molecular architecture of the link between cortical ER and the polarisome.


Assuntos
Proteínas de Transporte/química , Proteínas Ativadoras de GTPase/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Sequência de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
12.
Nucleic Acids Res ; 49(8): 4534-4549, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849072

RESUMO

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/metabolismo , Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
13.
Mol Genet Genomics ; 296(4): 905-918, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33914130

RESUMO

Phenotype is one of the most significant concepts in genetics, which is used to describe all the characteristics of a research object that can be observed. Considering that phenotype reflects the integrated features of genotype and environment factors, it is hard to define phenotype characteristics, even difficult to predict unknown phenotypes. Restricted by current biological techniques, it is still quite expensive and time-consuming to obtain sufficient structural information of large-scale phenotype-associated genes/proteins. Various bioinformatics methods have been presented to solve such problem, and researchers have confirmed the efficacy and prediction accuracy of functional network-based prediction. But general functional descriptions have highly complicated inner structures for phenotype prediction. To further address this issue and improve the efficacy of phenotype prediction on more than ten kinds of phenotypes, we first extract functional enrichment features from GO and KEGG, and then use node2vec to learn functional embedding features of genes from a gene-gene network. All these features are analyzed by some feature selection methods (Boruta, minimum redundancy maximum relevance) to generate a feature list. Such list is fed into the incremental feature selection, incorporating some multi-label classifiers built by RAkEL and some classic base classifiers, to build an optimum multi-label multi-class classification model for phenotype prediction. According to recent researches, our method has indeed identified many literature-supported genes/proteins and their associated phenotypes, and even some candidate genes with re-assigned new phenotypes, which provide a new computational tool for the accurate and effective phenotypic prediction.


Assuntos
Algoritmos , Biologia Computacional/métodos , Estudos de Associação Genética/métodos , Conjuntos de Dados como Assunto , Redes Reguladoras de Genes/fisiologia , Redes e Vias Metabólicas/genética , Fenótipo , Proteínas/química , Proteínas/genética , Proteínas/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Relação Estrutura-Atividade
14.
Methods Mol Biol ; 2263: 341-350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877606

RESUMO

Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.


Assuntos
Técnicas Biossensoriais/métodos , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fenômenos Biofísicos , Dicistroviridae/fisiologia , Sítios Internos de Entrada Ribossomal , Cinética , Modelos Moleculares , Estudo de Prova de Conceito , Biossíntese de Proteínas , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 12(1): 2370, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888705

RESUMO

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1-E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1-E2-Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1-E2-ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1-E2-Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Domínio Catalítico , Cristalografia por Raios X , Esterificação/fisiologia , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/isolamento & purificação
16.
Mol Cell ; 81(11): 2445-2459.e13, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33905682

RESUMO

How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Moléculas de Adesão Celular/química , Frutose-Bifosfatase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Microscopia Crioeletrônica , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Expressão Gênica , Gluconeogênese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
17.
Nucleic Acids Res ; 49(7): 3932-3947, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33772579

RESUMO

Palindromic sequences are a potent source of chromosomal instability in many organisms and are implicated in the pathogenesis of human diseases. In this study, we investigate which nucleases are responsible for cleavage of the hairpin and cruciform structures and generation of double-strand breaks at inverted repeats in Saccharomyces cerevisiae. We demonstrate that the involvement of structure-specific nucleases in palindrome fragility depends on the distance between inverted repeats and their transcriptional status. The attack by the Mre11 complex is constrained to hairpins with loops <9 nucleotides. This restriction is alleviated upon RPA depletion, indicating that RPA controls the stability and/or formation of secondary structures otherwise responsible for replication fork stalling and DSB formation. Mus81-Mms4 cleavage of cruciforms occurs at divergently but not convergently transcribed or nontranscribed repeats. Our study also reveals the third pathway for fragility at perfect and quasi-palindromes, which involves cruciform resolution during the G2 phase of the cell cycle.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Fúngico/metabolismo , Sequências Repetidas Invertidas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Conformação de Ácido Nucleico , Elementos Estruturais de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Phys Chem Lett ; 12(11): 2805-2808, 2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33710900

RESUMO

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.


Assuntos
Fosfoglicerato Quinase/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Histidina/genética , Histidina/imunologia , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
19.
Nat Commun ; 12(1): 1355, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649303

RESUMO

The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.


Assuntos
Sequência Conservada , Mamíferos/metabolismo , Complexo Mediador/química , Complexo Mediador/metabolismo , Animais , Linhagem Celular Tumoral , Doença/genética , Complexo Mediador/ultraestrutura , Camundongos , Modelos Moleculares , Mutação/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
20.
Acta Biochim Biophys Sin (Shanghai) ; 53(4): 463-471, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33751023

RESUMO

A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: α1-L1-ß1-L2-ß2-L3-ß3-L4-α2 (α represents an α-helix, ß a ß-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer's ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.


Assuntos
Motivo de Ligação ao RNA de Cadeia Dupla , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Células HEK293 , Humanos , MicroRNAs/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Estrutura Secundária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiologia
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