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1.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
2.
RNA ; 27(10): 1257-1264, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34257148

RESUMO

The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus, our Burkholderia structure reveals a chelated K+ ion adjacent to two Mg2+ ions in the guanidine-binding pocket. Thermal melting analysis shows that K+ chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K+(Mg2+)2] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of ykkC-I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K+ chelation in the Burkholderia Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg2+ ion binding in place of the K+ ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among ykkC-I riboswitches.


Assuntos
Burkholderia/genética , Quelantes/química , Guanidinas/química , Magnésio/química , Potássio/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Pareamento de Bases , Sequência de Bases , Evolução Biológica , Burkholderia/metabolismo , Quelantes/metabolismo , Clostridiales/genética , Clostridiales/metabolismo , Cristalografia por Raios X , Dickeya/genética , Dickeya/metabolismo , Guanidinas/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Potássio/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Água/química , Água/metabolismo
3.
RNA ; 27(9): 971-980, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34193550

RESUMO

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases.


Assuntos
Imunomodulação/efeitos dos fármacos , Nanoestruturas/química , Nanotecnologia/métodos , Oligodesoxirribonucleotídeos/farmacologia , Riboswitch , Animais , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Engenharia Genética/métodos , Concentração de Íons de Hidrogênio , Interleucina-6/biossíntese , Interleucina-6/imunologia , Ligantes , Camundongos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia
4.
Nucleic Acids Res ; 49(13): 7753-7764, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34223902

RESUMO

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1-D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical ß2-ß3-ß5 interfaces. Evidently, the 3'-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.


Assuntos
Proteínas de Bactérias/química , RNA Mensageiro/química , Proteínas Ribossômicas/química , Vibrio vulnificus/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , Riboswitch
5.
J Phys Chem B ; 125(30): 8342-8350, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34310879

RESUMO

Estimating the binding energies of small molecules to RNA could help uncover their molecular recognition characteristics and be used to rationally design RNA-targeting chemical probes. Here, we leveraged the ability of the fragment molecular orbital (FMO) method to provide detailed pairwise energetic information to examine the interactions between the aptamer domain of the flavin mononucleotide (FMN)-responsive riboswitch and small-molecule ligands. After developing an efficient protocol for executing high-level FMO calculations on RNA-ligand complexes, we applied our protocol to nine FMN-aptamer-ligand complexes. We then used the results to identify "hot-spots" within the aptamer and decomposed pairwise interactions between the hot-spot residues and the ligands. Interestingly, we found that several of these hot-spot residues interact with the ligands via atypical CH···O hydrogen bonds and anion-π contacts, as well as (face-to-edge) T-shaped π-π interactions. We envision that our results should pave the way for the wider and more prominent use of FMO calculations to study structure-energy relationships in diverse RNA-ligand systems, which in turn may provide a basis for dissecting the molecular recognition characteristics of RNAs.


Assuntos
Riboswitch , Mononucleotídeo de Flavina , Ligação de Hidrogênio , Ligantes , Conformação de Ácido Nucleico , RNA
6.
ACS Chem Biol ; 16(7): 1111-1127, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34166593

RESUMO

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.


Assuntos
Compostos Orgânicos/farmacologia , RNA/metabolismo , Animais , Linhagem Celular Tumoral , Descoberta de Drogas , Humanos , Splicing de RNA/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia
7.
Nucleic Acids Res ; 49(12): 7139-7153, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125892

RESUMO

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson-Crick base pair to junction J2. Xanthine inserts between a G-U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.


Assuntos
Magnésio/química , Riboswitch , Xantina/química , Sítios de Ligação , Cátions Bivalentes , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Dobramento de RNA
8.
J Phys Chem B ; 125(24): 6479-6490, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34106719

RESUMO

Magnesium plays a critical role in the structure, dynamics, and function of RNA. The precise microscopic effect of chelated magnesium on RNA structure is yet to be explored. Magnesium is known to act through its diffuse cloud around RNA, through the outer sphere (water-mediated), inner sphere, and often chelated ion-mediated interactions. A mechanism is proposed for the role of experimentally discovered site-specific chelated magnesium ions on the conformational dynamics of SAM-I riboswitch aptamers in bacteria. This mechanism is observed with atomistic simulations performed in a physiological mixed salt environment at a high temperature. The simulations were validated with phosphorothioate interference mapping experiments that help to identify crucial inner-sphere Mg2+ sites prescribing an appropriate initial distribution of inner- and outer-sphere magnesium ions to maintain a physiological ion concentration of monovalent and divalent salts. A concerted role of two chelated magnesium ions is newly discovered since the presence of both supports the formation of the pseudoknot. This constitutes a logical AND gate. The absence of any of these magnesium ions instigates the dissociation of long-range pseudoknot interaction exposing the inner core of the RNA. A base triple is the epicenter of the magnesium chelation effect. It allosterically controls RNA pseudoknot by bolstering the direct effect of magnesium chelation in protecting the functional fold of RNA to control ON and OFF transcription switching.


Assuntos
Riboswitch , Magnésio , Conformação de Ácido Nucleico , RNA/genética
9.
Methods Mol Biol ; 2323: 121-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086278

RESUMO

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.


Assuntos
Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , RNA de Transferência/genética , RNA/genética , Riboswitch/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos de Benzil , Clonagem Molecular/métodos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imidazolinas , Isopropiltiogalactosídeo/farmacologia , Conformação de Ácido Nucleico , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Plasmídeos
10.
Methods Mol Biol ; 2323: 153-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086280

RESUMO

Riboswitches are a class of noncoding RNAs that regulate gene expression in response to changes in intracellular metabolite concentrations. When riboswitches are placed upstream of genetic reporters, the degree of reporter activity reflects the relative abundance of the metabolite that is sensed by the riboswitch. This method describes how reporters for live cell imaging, such as yellow fluorescent protein (YFP), can be placed under genetic control by metabolite-sensing riboswitches in the bacterium Bacillus subtilis. Specifically, a protocol for generating a fluorescent YFP reporter, based on a c-di-GMP responsive riboswitch, is outlined below.


Assuntos
Proteínas de Bactérias/análise , Genes Reporter , Microscopia Intravital/métodos , Proteínas Luminescentes/análise , Riboswitch/genética , Regiões 5' não Traduzidas/genética , Aptâmeros de Nucleotídeos/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Regulação Bacteriana da Expressão Gênica , Ligantes , Proteínas Luminescentes/genética , Conformação de Ácido Nucleico , Plasmídeos , Transformação Bacteriana
11.
Methods Mol Biol ; 2323: 199-212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086282

RESUMO

Artificial RNA switches are an emerging class of genetic controllers suitable for synthetic biology applications. Aptazymes are fusions composed of an aptamer domain and a self-cleaving ribozyme. The utilization of aptazymes for conditional gene expression displays several advantages over employing conventional transcription factor-based techniques as aptazymes require minimal genomic space, fulfill their function without the need of protein cofactors and most importantly are reprogrammable with respect to ligand selectivity and the RNA function to be regulated. Technologies that enable the generation of aptazymes to defined input ligands are of interest for the construction of biocomputing devices and biosensing applications. In this chapter we present a method that facilitates the in vivo screening of randomized pools of aptazymes in mammalian cells.


Assuntos
Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Engenharia Genética/métodos , RNA Catalítico/genética , RNA/genética , Riboswitch/genética , Animais , Computadores Moleculares , Biblioteca Gênica , Genes Reporter , Ligantes , Mamíferos/genética , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA/metabolismo , Especificidade por Substrato
12.
Methods Mol Biol ; 2323: 213-220, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086283

RESUMO

This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3' untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3' UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression.


Assuntos
Aptâmeros de Nucleotídeos , Tecnologia Digital/métodos , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Engenharia Genética/métodos , Lógica , Riboswitch , Regiões 3' não Traduzidas/genética , Regulação Alostérica , Animais , Sequência de Bases , Clonagem Molecular/métodos , Biblioteca Gênica , Células HEK293 , Humanos , Mamíferos , Plasmídeos/genética , RNA Catalítico , Transfecção
13.
Nucleic Acids Res ; 49(11): 6128-6143, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086938

RESUMO

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3' UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.


Assuntos
RNA Fúngico/química , RNA não Traduzido/química , Regiões 3' não Traduzidas , Biologia Computacional/métodos , Genoma Fúngico , Íntrons , Lisina-tRNA Ligase/genética , Cadeias de Markov , Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/química , Proteínas Ribossômicas/genética , Riboswitch , Alinhamento de Sequência , Tiorredoxinas/genética
14.
Curr Microbiol ; 78(8): 2943-2955, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34076709

RESUMO

The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.


Assuntos
Aptâmeros de Nucleotídeos , Burkholderia , Riboswitch , Burkholderia/genética , Glicina/genética , Ligantes , Conformação de Ácido Nucleico , Riboswitch/genética
15.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
16.
Methods Mol Biol ; 2317: 305-318, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028778

RESUMO

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Riboswitch
17.
Nucleic Acids Res ; 49(10): 5891-5904, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33963862

RESUMO

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.


Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Vibrio vulnificus/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Dobramento de RNA , Riboswitch , Imagem Individual de Molécula , Software , Espectroscopia de Infravermelho com Transformada de Fourier , Vibrio vulnificus/genética
18.
Nucleic Acids Res ; 49(12): e71, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-33893804

RESUMO

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.


Assuntos
Processamento Alternativo , Caenorhabditis elegans/genética , Riboswitch , Tetraciclina/farmacologia , Processamento Alternativo/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos , Éxons , Expressão Gênica , Células HeLa , Humanos
19.
J Phys Chem B ; 125(14): 3486-3493, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33818089

RESUMO

Riboswitches are regulatory ribonucleic acid (RNA) elements that act as ligand-dependent conformational switches that recognize their cognate ligand via a binding pocket located in their aptamer domain. In the apo form, the aptamer domain is dynamic, requiring an ensemble representation of its structure. Here, as a proof-of-concept, we used solvent accessibility information to construct a pair of dynamical ensembles of the aptamer domain of the well-studied S-adenosylmethionine (SAM) class-I riboswitch in the absence (-SAM) and presence (+SAM) of SAM. To achieve this, we first generated a large conformational library and then reweighted conformers in the library using solvent-accessible surface area (SASA) data derived from recently reported light-activated structural examination of RNA (LASER) reactivities measured in the -SAM and +SAM states of the riboswitch. The differences in the resulting -SAM and +SAM ensembles are consistent with a SAM-dependent reshaping of the free-energy landscape of the aptamer domain. Within our -SAM ensemble, we identified a "transient" state that is missing a critical long-range contact, leading us to speculate that it may be representative of a folding intermediate. Further structural analysis also revealed that the transient state harbors a hidden binding pocket that is distinct from the SAM-binding pocket and is predicted by docking calculations to selectively bind small-molecule ligands. The SASA-based method we applied to the SAM-I riboswitch aptamer domain is general and could be used to construct dynamical ensembles for other riboswitch aptamer domains and, more broadly, other classes of structured RNAs.


Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Ligantes , Conformação de Ácido Nucleico , RNA , S-Adenosilmetionina , Solventes
20.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810132

RESUMO

Riboswitches are segments of noncoding RNA that bind with metabolites, resulting in a change in gene expression. To understand the molecular mechanism of gene regulation in a fluoride riboswitch, a base-pair opening dynamics study was performed with and without ligands using the Bacillus cereus fluoride riboswitch. We demonstrate that the structural stability of the fluoride riboswitch is caused by two steps depending on ligands. Upon binding of a magnesium ion, significant changes in a conformation of the riboswitch occur, resulting in the greatest increase in their stability and changes in dynamics by a fluoride ion. Examining hydrogen exchange dynamics through NMR spectroscopy, we reveal that the stabilization of the U45·A37 base-pair due to the binding of the fluoride ion, by changing the dynamics while maintaining the structure, results in transcription regulation. Our results demonstrate that the opening dynamics and stabilities of a fluoride riboswitch in different ion states are essential for the genetic switching mechanism.


Assuntos
Bacillus cereus/genética , Pareamento de Bases , Fluoretos/química , Genes Bacterianos , Riboswitch , Aptâmeros de Nucleotídeos , Sequência de Bases , Catálise , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos
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