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1.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
2.
Chem Sci ; 9(36): 7271-7276, 2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30288248

RESUMO

Nanoscale engineering of surfaces is becoming an indispensable technique to modify membranes and, thus cellular behaviour. Here, such membrane engineering related was explored on the surface of a living animal using DNA nanotechnology. We demonstrate the immobilization of oligonucleotides functionalized with a membrane anchor on 2 day old zebrafish. The protruding single-stranded DNA on the skin of zebrafish served as a handle for complementary DNAs, which allowed the attachment of small molecule cargo, liposomes and dynamic relabeling by DNA hybridization protocols. Robust anchoring of the oligonucleotides was proven as DNA-based amplification processes were successfully performed on the outer membrane of the zebrafish enabling the multiplication of surface functionalities from a single DNA-anchoring unit and the dramatic improvement of fluorescent labeling of these animals. As zebrafish are becoming an alternative to animal models in drug development, toxicology and nanoparticles characterization, we believe the platform presented here allows amalgamation of DNA nanotechnology tools with live animals and this opens up yet unexplored avenues like efficient bio-barcoding as well as in vivo tracking.

3.
Adv Healthc Mater ; 6(20)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28945015

RESUMO

Protein delivery into the cytosol of cells is a challenging topic in the field of nanomedicine, because cellular uptake and endosomal escape are typically inefficient, hampering clinical applications. In this contribution cuboidal mesoporous silica nanoparticles (MSNs) containing disk-shaped cavities with a large pore diameter (10 nm) are studied as a protein delivery vehicle using cytochrome-c (cytC) as a model membrane-impermeable protein. To ensure colloidal stability, the MSNs are coated with a fusogenic lipid bilayer (LB) and cellular uptake is induced by a complementary pair of coiled-coil (CC) lipopeptides. Coiled-coil induced membrane fusion leads to the efficient cytosolic delivery of cytC and triggers apoptosis of cells. Delivery of these LB coated MSNs in the presence of various endocytosis inhibitors strongly suggests that membrane fusion is the dominant mechanism of cellular uptake. This method is potentially a universal way for the efficient delivery of any type of inorganic nanoparticle or protein into cells mediated by CC induced membrane fusion.


Assuntos
Materiais Revestidos Biocompatíveis/química , Bicamadas Lipídicas/química , Nanopartículas/química , Dióxido de Silício/química , Apoptose/efeitos dos fármacos , Citocromos c/química , Citocromos c/metabolismo , Citocromos c/toxicidade , Citosol/metabolismo , Endocitose , Células HeLa , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Fusão de Membrana , Microscopia Confocal , Tamanho da Partícula , Porosidade
4.
Sci Rep ; 6: 38892, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27966593

RESUMO

The influenza A virus genome consists of eight RNA segments. RNA structures within these segments and complementary (cRNA) and protein-coding mRNAs may play a role in virus replication. Here, conserved putative secondary structures that impose significant evolutionary constraints on the gene segment encoding the surface glycoprotein hemagglutinin (HA) were investigated using available sequence data on tens of thousands of virus strains. Structural constraints were identified by analysis of covariations of nucleotides suggested to be paired by structure prediction algorithms. The significance of covariations was estimated by mutual information calculations and tracing multiple covariation events during virus evolution. Covariation patterns demonstrated that structured domains in HA RNAs were mostly subtype-specific, whereas some structures were conserved in several subtypes. The influence of RNA folding on virus replication was studied by plaque assays of mutant viruses with disrupted structures. The results suggest that over the whole length of the HA segment there are local structured domains which contribute to the virus fitness but individually are not essential for the virus. Existence of subtype-specific structured regions in the segments of the influenza A virus genome is apparently an important factor in virus evolution and reassortment of its genes.


Assuntos
Evolução Molecular , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética
5.
Sci Rep ; 6: 39549, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28000744

RESUMO

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Produtos do Gene gag/química , RNA Viral/genética , Retrovirus dos Símios , Ribossomos/química , Eletroforese em Gel de Poliacrilamida , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Pinças Ópticas , Desnaturação Proteica , Dobramento de Proteína , RNA Helicases/química , RNA Mensageiro/metabolismo , Termodinâmica
6.
ACS Cent Sci ; 2(9): 621-630, 2016 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-27725960

RESUMO

Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and invivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivo applications.

7.
ACS Nano ; 10(8): 7428-35, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27504667

RESUMO

The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery.


Assuntos
Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Xenoenxertos , Lipossomos , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Peptídeos , Peixe-Zebra
8.
Methods Enzymol ; 550: 385-93, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25605396

RESUMO

Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Riboswitch/genética , RNA Bacteriano/genética
9.
RNA Biol ; 11(7): 942-52, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25180940

RESUMO

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.


Assuntos
Vírus da Influenza A/fisiologia , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Animais , Cães , Evolução Molecular , Células HEK293 , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação , Dobramento de RNA , RNA Viral/genética , Montagem de Vírus
10.
Virus Res ; 190: 110-7, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-25051146

RESUMO

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element.


Assuntos
Regiões 3' não Traduzidas , Regulação Viral da Expressão Gênica , Potexvirus/genética , RNA Viral/genética , Sequência de Bases , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Doenças das Plantas/virologia , Potexvirus/metabolismo , RNA Viral/química , RNA Viral/metabolismo
11.
Nucleic Acids Res ; 42(14): 9327-33, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25030900

RESUMO

Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrP(C) to infectious PrP(Sc) or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrP(C) have been isolated and found to form G-quadruplexes (G4s). PrP(C) binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrP(Sc). Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed.


Assuntos
Quadruplex G , Príons/genética , RNA Mensageiro/química , Humanos , Oligorribonucleotídeos/química , Doenças Priônicas/genética , Biossíntese de Proteínas
12.
Virol J ; 11: 116, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24946926

RESUMO

BACKGROUND: RNA bacteriophages like Qbeta and MS2 are well known for their high mutation rate, short infection cycle and strong selection against foreign inserts. The hammerhead ribozyme (HHRz) is a small self-cleaving RNA molecule whose active residues have previously been identified by mutational analysis of each individual base. Here the functionally important bases of HHRz were determined in a single screening experiment by inserting the HHRz into the genome of MS2. FINDINGS: The minimal HHRz of satellite Tobacco ringspot virus was cloned into the genome of RNA bacteriophage MS2. Sequence analysis of the surviving phages revealed that the majority had acquired single base-substitutions that apparently inactivated the HHRz. The positions of these substitutions exactly matched that of the previously determined core residues of the HHRz. CONCLUSIONS: Natural selection against a ribozyme in the genome of MS2 can be used to quickly identify nucleotides required for self-cleavage.


Assuntos
Levivirus/genética , Nepovirus/enzimologia , RNA Catalítico/genética , RNA Catalítico/metabolismo , Análise Mutacional de DNA , Levivirus/enzimologia , Nepovirus/genética , Nucleotídeos , Mutação Puntual , RNA/genética , RNA/metabolismo , Seleção Genética
13.
Bioinformatics ; 30(13): 1800-4, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24590440

RESUMO

The intergenic regions of the ambisense RNA segments of viruses from the Tospovirus genus form large extended RNA structures that regulate virus replication. Using comparative structure analysis, we show the presence of conserved alternative conformations at the apical parts of these structures. In one conformation, a branched Y-shape, the 5'-proximal hairpin arms are mostly capped by exceptionally stable tetraloop motifs. The tetraloop hairpins are folded in both virus and virus-complementary sense RNAs, and different tetraloops can functionally replace each other. Folding simulations show that the branched Y-shape structures can undergo a conformational transition to alternative extended rod-like conformations. Functional importance of both alternatives is supported by nucleotide covariations. The balanced equilibrium between alternative structures is evidenced by native gel electrophoresis of mutant RNA transcripts with shifted equilibria. The tetraloops play a role in the stability and dynamics of structures but may also be recognized by proteins involved in translation and/or replication.


Assuntos
DNA Intergênico , Genoma Viral , RNA Viral/química , Tospovirus/química , Genômica , Conformação de Ácido Nucleico , RNA Viral/genética , Tospovirus/genética
14.
Nucleic Acids Res ; 42(3): 1887-92, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24178029

RESUMO

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating -1 ribosomal frameshifting (-1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the -1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher -1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Quadruplex G , RNA/química , Regiões 5' não Traduzidas , Códon de Terminação , Ligantes , RNA/metabolismo , Termodinâmica
15.
RNA ; 19(12): 1833-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24158793

RESUMO

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross-loop base pair leaving a triloop and 3' bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross-loop base pair, also a 3' pyrimidine bulge and a G-C loop-closing base pair are primary determinants of PTL formation. By changing the G-C closing base pair into C-G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.


Assuntos
RNA Viral/química , Pareamento de Bases , Vírus da Hepatite B/genética , Sequências Repetidas Invertidas , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Estabilidade de RNA/efeitos da radiação , Vírus Espumoso dos Símios/genética , Termodinâmica , Raios Ultravioleta
16.
ACS Chem Biol ; 8(4): 733-40, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23327288

RESUMO

Knowing the molecular details of the interaction between riboswitch aptamers and their corresponding metabolites is important to understand gene expression. Here we report on a novel in vitro assay to study preQ(1) riboswitch aptamers upon binding of 7-aminomethyl-7-deazaguanine (preQ(1)). The assay is based on the ability of the preQ(1) aptamer to fold, upon ligand binding, into a pseudoknotted structure that is capable of stimulating -1 ribosomal frameshifting (-1 FS). Aptamers from three different species were found to induce between 7% and 20% of -1 FS in response to increasing preQ(1) levels, whereas preQ(1) analogues were 100-1000-fold less efficient. In depth mutational analysis of the Fusobacterium nucleatum aptamer recapitulates most of the structural details previously identified for preQ(1) aptamers from other bacteria by crystallography and/or NMR spectroscopy. In addition to providing insight into the role of individual nucleotides of the preQ(1) riboswitch aptamer in ligand binding, the presented system provides a valuable tool to screen small molecules against bacterial riboswitches in a eukaryotic background.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Riboswitch , Aptâmeros de Nucleotídeos , Sequência de Bases , Conformação de Ácido Nucleico
17.
Virus Res ; 167(2): 267-72, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22617023

RESUMO

Pepino mosaic virus (PepMV)-infected tomato plants were used to develop an in vitro template-dependent system for the study of viral RNA synthesis. Differential sedimentation and sucrose-gradient purification of PepMV-infected tomato extracts resulted in fractions containing a transcriptionally active membrane-bound RNA-dependent RNA polymerase (RdRp). In the presence of Mg(2+) ions, (32)P-labelled UTP and unlabelled ATP, CTP, GTP, the PepMV RdRp catalysed the conversion of endogenous RNA templates into single- and double-stranded (ds) genomic RNAs and three 3'-co-terminal subgenomic dsRNAs. Hybridisation experiments showed that the genomic ssRNA was labelled only in the plus strand, the genomic dsRNA mainly in the plus strand and the three subgenomic dsRNAs equally in both strands. Following removal of the endogenous templates from the membrane-bound complex, the purified template-dependent RdRp could specifically catalyse transcription of PepMV virion RNA, in vitro-synthesized full-length plus-strand RNA and the 3'-termini of both the plus- and minus-strand RNAs. Rabbit polyclonal antibodies against an immunogenic epitope of the PepMV RdRp (anti-RdRp) detected a protein of approximately 164kDa in the membrane-bound and template-dependent RdRp preparations and exclusively inhibited PepMV RNA synthesis when added to the template-dependent in vitro transcription system. The 300 nucleotides long 3'-terminal region of the PepMV genome, containing a stretch of at least 20 adenosine (A) residues, was an adequate exogenous RNA template for RdRp initiation of the minus-strand synthesis but higher transcription efficiency was observed as the number of A residues increased. This observation might indicate a role for the poly(A)-tail in the formation and stabilisation of secondary structure(s) essential for initiation of transcription. The template-dependent specific RdRp system described in this article will facilitate identification of RNA elements and host components required for PepMV RNA synthesis.


Assuntos
Lycopersicon esculentum/virologia , Potexvirus/enzimologia , Potexvirus/genética , RNA Replicase/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Coenzimas/metabolismo , Magnésio/metabolismo , Extratos Vegetais/metabolismo
18.
RNA ; 18(5): 992-1000, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22393035

RESUMO

In brome mosaic virus, both the replication of the genomic (+)-RNA strands and the transcription of the subgenomic RNA are carried out by the viral replicase. The production of (-)-RNA strands is dependent on the formation of an AUA triloop in the stem-loop C (SLC) hairpin in the 3'-untranslated region of the (+)-RNA strands. Two alternate hypotheses have been put forward for the mechanism of subgenomic RNA transcription. One posits that transcription commences by recognition of at least four key nucleotides in the subgenomic promoter by the replicase. The other posits that subgenomic transcription starts by binding of the replicase to a hairpin formed by the subgenomic promoter that resembles the minus strand promoter hairpin SLC. In this study, we have determined the three-dimensional structure of the subgenomic promoter hairpin using NMR spectroscopy. The data show that the hairpin is stable at 30°C and that it forms a pseudo-triloop structure with a transloop base pair and a nucleotide completely excluded from the helix. The transloop base pair is capped by an AUA triloop that possesses an extremely well packed structure very similar to that of the AUA triloop of SLC, including the formation of a so-called clamped-adenine motif. The similarities of the NMR structures of the hairpins required for genomic RNA and subgenomic RNA synthesis show that the replicase recognizes structure rather than sequence-specific motifs in both promoters.


Assuntos
Bromovirus/genética , Genoma Viral , Sequências Repetidas Invertidas , Regiões Promotoras Genéticas , RNA Viral/química , Pareamento de Bases , Sequência de Bases , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Termodinâmica
19.
Chemphyschem ; 13(6): 1569-75, 2012 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-22407519

RESUMO

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration.


Assuntos
DNA/química , Nanotubos de Carbono/química , Proteínas/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química
20.
Nucleic Acids Res ; 39(20): 8952-9, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21803791

RESUMO

-1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base-base and base-sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in -1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol/genética , RNA Mensageiro/química , RNA Viral/química , Composição de Bases , Pareamento Incorreto de Bases , Células HeLa , Humanos , Vírus dos Macacos de Mason-Pfizer/genética , Conformação de Ácido Nucleico
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