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A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. 1H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (â¼5-6%) ACSL conformations. Exchange rate constant (kex) for interconversion between P and S states is 112 sec-1 (<Δω â¼454 rad/sec), confirming a slow exchange regime between the two states. Forward (kPS) and backward (kSP) rate constants are 49.166 sec-1 and 62.792 sec-1, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by 1H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs.Communicated by Ramaswamy H. Sarma.
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Nucleotídeos , RNA , RNA/genética , Espectroscopia de Ressonância Magnética , RNA de Transferência , Anticódon , Conformação de Ácido NucleicoRESUMO
Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila, six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder.
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With recent emergence of huge number of long noncoding RNAs (lncRNAs), purification of lncRNA-protein (lncRNP) complexes is fundamental to understand the role of lncRNA and its biological function. However, lncRNP purification is still a daunting challenge. Here we describe a protocol to purify lncRNP formed in vivo with MS2-MBP-based affinity purification. Inducible lncRNA tagged with MS2 RNA hairpins is introduced into cells of interest, and RNP on tagged lncRNA is formed in vivo. MS2-MBP fusion protein is expressed in Escherichia coli and purified with amylose resin and HiTrap heparin column. The MS2 part of MS2-MBP fusion protein binds to the hairpins, and MBP part binds to amylose resin. We also describe a protocol to separate the nucleus and the cytoplasm so that lncRNP localized in the nucleus or cytoplasm can be individually purified. The amount of lncRNP purified is well sufficient for mass spectrometry analysis.
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RNA Longo não Codificante , RNA Longo não Codificante/metabolismo , Amilose , Cromatografia de Afinidade/métodos , Indicadores e Reagentes , Núcleo Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Ligantes de MaltoseRESUMO
Advances in single-molecule experiments on macromolecular crowding urgently need an efficient simulation method to resolve their discrepancies quantitatively. Ox-DNA model has been since reworked to treat the thermodynamics and mechanical properties of DNA/RNA hairpin at a stretching force. In hopping experiments, the critical forces of RNA hairpins at different temperatures are greater than those of DNA hairpins, in addition, the Gibbs free energy at a fixed temperature required to convert an RNA hairpin into a single-stranded molecule at zero force is obviously greater than that of DNA hairpin and gradually decreases by increasing the temperature. As far as force-ramping experiments are concerned, the first-rupture forces of RNA/DNA hairpins corresponding to the maximum probability density linearly pertain to the force-loading rate, with those of RNA hairpins being greater. The extended ox-DNA model could potentially identify the interaction between biologically inert polymer and RNA/DNA hairpins in crowded environments.
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DNA , RNA , Conformação de Ácido Nucleico , Temperatura , TermodinâmicaRESUMO
Pseudoknotted RNA molecules play important biological roles that depend on their folded structure. To understand the underlying principles that determine their thermodynamics and folding/unfolding mechanisms, we carried out a study on a variant of the mouse mammary tumor virus pseudoknotted RNA (VPK), a widely studied model system for RNA pseudoknots. Our method is based on a coarse-grained discrete-state model and the algorithm of PK3D (pseudoknot structure predictor in three-dimensional space), with RNA loops explicitly constructed and their conformational entropic effects incorporated. Our loop entropy calculations are validated by accurately capturing previously measured melting temperatures of RNA hairpins with varying loop lengths. For each of the hairpins that constitutes the VPK, we identified alternative conformations that are more stable than the hairpin structures at low temperatures and predicted their populations at different temperatures. Our predictions were validated by thermodynamic experiments on these hairpins. We further computed the heat capacity profiles of VPK, which are in excellent agreement with available experimental data. Notably, our model provides detailed information on the unfolding mechanisms of pseudoknotted RNA. Analysis of the distribution of base-pairing probability of VPK reveals a cooperative unfolding mechanism instead of a simple sequential unfolding of first one stem and then the other. Specifically, we find a simultaneous "loosening" of both stems as the temperature is raised, whereby both stems become partially melted and co-exist during the unfolding process.
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Vírus do Tumor Mamário do Camundongo , RNA , Animais , Entropia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Conformação de Ácido Nucleico , RNA/química , TermodinâmicaRESUMO
Huntington's disease (HD) is caused by a CAG-repeat expansion mutation in the Huntingtin (HTT) gene. It is characterized by progressive psychiatric and neurological symptoms in combination with a progressive movement disorder. Despite the ubiquitous expression of HTT, pathological changes occur quite selectively in the central nervous system. Since the discovery of HD more than 150 years ago, a lot of research on molecular mechanisms contributing to neurotoxicity has remained the focal point. While traditionally, the protein encoded by the HTT gene remained the cynosure for researchers and was extensively reviewed elsewhere, several studies in the last few years clearly indicated the contribution of the mutant RNA transcript to cellular dysfunction as well. In this review, we outline recent studies on RNA-mediated molecular mechanisms that are linked to cellular dysfunction in HD models. These mechanisms include mis-splicing, aberrant translation, deregulation of the miRNA machinery, deregulated RNA transport and abnormal regulation of mitochondrial RNA. Furthermore, we summarize recent therapeutical approaches targeting the mutant HTT transcript. While currently available treatments are of a palliative nature only and do not halt the disease progression, recent clinical studies provide hope that these novel RNA-targeting strategies will lead to better therapeutic approaches.
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Proteína Huntingtina/metabolismo , MicroRNAs , Animais , Doença de Huntington/genética , MutaçãoRESUMO
Biosensors are of interest for the quantitative detection of small molecules (metabolites, drugs and contaminants for instance). To this end, fluorescence is a widely used technique that is easily associated to aptamers. Light-up aptamers constitute a particular class of oligonucleotides that, specifically induce fluorescence emission when binding to cognate fluorogenic ligands such as malachite green (MG). We engineered a dual aptasensor for theophylline (Th) based on the combination of switching hairpin aptamers specific for MG on the one hand and for Th on the other hand, hence their names: malaswitch (Msw) and theoswitch (Thsw). The two aptaswitches form a loop-loop or kissing Msw-Thsw complex only in the presence of theophylline, allowing binding of MG, subsequently generating a fluorescent signal. The combination of the best Msw and Thsw variants, MswG12 and Thsw19.1, results in a 20-fold fluorescence enhancement of MG at saturating theophylline concentration. This aptasensor discriminates between theophylline and its analogues caffeine and theobromine. Kissing aptaswitches derived from light-up aptamers constitute a novel sensing device.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Corantes de Rosanilina , TeofilinaRESUMO
Nucleotide modification in RNA controls a bevy of biological processes, including RNA degradation, gene expression, and gene editing. In turn, misregulation of modified nucleotides is associated with a host of chronic diseases and disorders. However, the molecular mechanisms driving these processes remain poorly understood. To partially address this knowledge gap, we used alchemical and temperature replica exchange molecular dynamics (TREMD) simulations on an RNA duplex and an analogous hairpin to probe the structural effects of modified and/or mutant nucleotides. The simulations successfully predict the modification/mutation-induced relative free energy change for complementary duplex formation, and structural analyses highlight mechanisms driving stability changes. Furthermore, TREMD simulations for a hairpin-forming RNA with and without modification provide reliable estimations of the energy landscape. Illuminating the impact of methylated and/or mutated nucleotides on the structure-function relationship and the folding energy landscape, the simulations provide insights into modification-induced alterations to the folding mechanics of the hairpin. The results here may be biologically significant as hairpins are widespread structure motifs that play critical roles in gene expression and regulation. Specifically, the tetraloop of the probed hairpin is phylogenetically abundant, and the stem mirrors a miRNA seed region whose modification has been implicated in epilepsy pathogenesis.
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Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química , Estabilidade de RNA , RNA/química , Termodinâmica , Nucleotídeos/genética , RNA/genética , TemperaturaRESUMO
The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read techniques have complementary strengths to overcome these limitations, with a particular strength in identifying structural variants. By using optical genome mapping and long-read sequencing, we aimed to identify the pathogenic variant in a large family with X-linked choroideremia. In this family, aberrant splicing of exon 12 of the choroideremia gene CHM was detected in 2003, but the underlying genomic defect remained elusive. Optical genome mapping and long-read sequencing approaches now revealed an intragenic 1,752 bp inverted duplication including exon 12 and surrounding regions, located downstream of the wild-type copy of exon 12. Both breakpoint junctions were confirmed with Sanger sequencing and segregate with the X-linked inheritance in the family. The breakpoint junctions displayed sequence microhomology suggestive for an erroneous replication mechanism as the origin of the structural variant. The inverted duplication is predicted to result in a hairpin formation of the pre-mRNA with the wild-type exon 12, leading to exon skipping in the mature mRNA. The identified inverted duplication is deemed the hidden pathogenic cause of disease in this family. Our study shows that optical genome mapping and long-read sequencing have significant potential for the identification of (hidden) structural variants in rare genetic diseases.
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I analyzed the model that suggests that an RNA ring might have been the progenitor of the tRNA molecule (Demongeot and Moreira in J Theor Biol 249:314-324, 2007; Demongeot and Seligmann in J Mol Evol 1-23, 2019a; Demongeot and Norris in Life 9(2):51, 2019). In particular, I analyze three ways in which this precursor, especially in its RNA hairpin form, could have evolved into the complete tRNA molecule. These three modalities are based on multiple duplication events, and therefore, appear to be less parsimonious than that which assumes that this molecule originated through one duplication of a single hairpin structure. The conclusion is, therefore, that the latter model appears to be preferable with respect to that of the RNA ring, also because there are many independent observations and some of a historical nature that would corroborate it in an extraordinary way.
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Evolução Molecular , Modelos Genéticos , Conformação de Ácido Nucleico , RNA de Transferência/genética , RNA/genética , Sequências Repetidas InvertidasRESUMO
Reliable conformational sampling and trajectory analysis are always important to the study of the folding or binding mechanisms of biomolecules. Generally, one has to prepare many complicated parameters and follow a lot of steps to obtain the final data. The whole process is too complicated to new users. In this article, we provide a convenient and user-friendly tool that is compatible to AMBER, called fast sampling and analysis tool (FSATOOL). FSATOOL has some useful features. First and the most important, the whole work is extremely simplified into two steps, one is the fast sampling procedure and the other is the trajectory analysis procedure. Second, it contains several powerful sampling methods for the simulation on graphics process unit, including our previous mixing replica exchange molecular dynamics method. The method combines the advantages of the biased and unbiased simulations. Finally, it extracts the dominant transition pathways automatically from the folding network by Markov state model. Users do not need to do the tedious intermediate steps by hand. To illustrate the usage of FSATOOL in practice, we perform one simulation for a RNA hairpin in explicit solvent. All the results are presented. © 2019 Wiley Periodicals, Inc.
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Simulação de Dinâmica Molecular , RNA/química , Conformação de Ácido Nucleico , Solventes/químicaRESUMO
RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82-148) NS RNA region influence NS1 protein expression.
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Expressão Gênica , Conformação de Ácido Nucleico , Orthomyxoviridae/crescimento & desenvolvimento , RNA Viral/química , Proteínas não Estruturais Virais/biossíntese , Animais , Cães , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Orthomyxoviridae/genética , Mutação Puntual , RNA Viral/metabolismo , Genética Reversa , Replicação ViralRESUMO
Cassava mosaic disease (CMD), caused by geminiviruses, is a major hurdle to cassava production. Due to the heterozygous nature of cassava, breeding for virus resistance is difficult, but cassava has been shown to be a good candidate for genetic engineering using RNA interference (RNAi). T This study reports on the ability of a transgene-derived RNA hairpin, homologous to an overlapping region of the SACMV replication associated protein and putative virus suppressor of silencing protein (AC1/AC4), to confer tolerance in the CMD-susceptible model cassava cultivar 60444. Three of the fourteen transgenic lines expressing SACMV AC1/AC4 hairpin-derived siRNAs showed decreased symptoms and viral loads compared to untransformed control plants. Expression of SACMV AC1/AC4 homologous siRNAs showed that this tolerance is most likely associated with post-transcriptional gene silencing of the virus. This is the first report of targeting the overlapping AC1 and AC4 genes of SACMV conferring CMD tolerance in cassava.
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Pausing by RNA polymerase (RNAP) during transcription regulates gene expression in all domains of life. In this review, we recap the history of transcriptional pausing discovery, summarize advances in our understanding of the underlying causes of pausing since then, and describe new insights into the pausing mechanisms and pause modulation by transcription factors gained from structural and biochemical experiments. The accumulated evidence to date suggests that upon encountering a pause signal in the nucleic-acid sequence being transcribed, RNAP rearranges into an elemental, catalytically inactive conformer unable to load NTP substrate. The conformation, and as a consequence lifetime, of an elemental paused RNAP is modulated by backtracking, nascent RNA structure, binding of transcription regulators, or a combination of these mechanisms. We conclude the review by outlining open questions and directions for future research in the field of transcriptional pausing.
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Bactérias/enzimologia , Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , DNA Bacteriano/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , Conformação de Ácido Nucleico , Conformação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Bacterial transcription termination, described mostly for Escherichia coli, occurs in three recognized ways: intrinsic termination, an activity only of the core RNAP enzyme and transcript sequences that encode an RNA hairpin and terminal uridine-rich segment; termination by the enzyme Rho, an ATP-dependent RNA translocase that releases RNA by forcing uncharacterized structural changes in the elongating complex; and Mfd-dependent termination, the activity of an ATP-dependent DNA translocase that is thought to dissociate the elongation complex by exerting torque on a stalled RNAP. Intrinsic termination can be described in terms of the nucleic acid movements in the process, whereas the enzymatic mechanisms have been illuminated importantly by definitive structural and biochemical analysis of their activity.
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RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Terminação da Transcrição Genética , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/metabolismo , Fator Rho/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG-repeat expansion in the 5' UTR of the FMR1 gene on the X-chromosome. Both elevated levels of the expanded FMR1 mRNA and aberrant expression of a polyglycine protein (FMRpolyG) from the CGG-repeat region are hypothesized to trigger the pathogenesis of FXTAS. While increased expression of FMRpolyG leads to higher toxicity in FXTAS models, the pathogenic effect of this protein has only been studied in the presence of CGG-containing mRNA. Here we present a model that allows measurement of the effect of FMRpolyG-expression without co-expression of the corresponding CGG mRNA hairpin. This allows direct comparison of the effect of the FMRpolyG protein per se, vs. that of the FMRpolyG protein together with the CGG mRNA hairpin. Our results show that expression of the FMRpolyG, in the absence of any CGG mRNA, is sufficient to cause reduced cell viability, lamin ring disruption and aggregate formation. Furthermore, we found FMRpolyG to be a long-lived protein degraded primarily by the ubiquitin-proteasome-system. Together, our data indicate that accumulation of FMRpolyG protein per se may play a major role in the development of FXTAS.
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Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.
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Fator Proteico 1 do Hospedeiro/metabolismo , Sequências Repetidas Invertidas , RNA/genética , RNA/metabolismo , Regiões Terminadoras Genéticas , Regiões 3' não Traduzidas , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Fosfatos/metabolismo , Poli U , Ligação Proteica , RNA/química , Estabilidade de RNA , RNA Bacteriano , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico , Transcrição GênicaRESUMO
The expansion of CAG repeats has been found to be associated with at least nine human genetic disorders. In these disorders, the full-length expanded CAG RNA transcripts are cleaved into small CAG-repeated RNAs which are cytotoxic and known to be capable of forming hairpins. To better understand the RNA pathogenic mechanism, in this study we have performed high-resolution nuclear magnetic resonance structural investigations on the RNA hairpins formed by CAG repeats. Our results show the formation of a type III AGCA tetraloop and reveal the effect of stem rigidity on the loop conformational flexibility.
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Modelos Moleculares , RNA/química , Pareamento Incorreto de Bases , Pareamento de Bases , Ligação de Hidrogênio , Sequências Repetidas Invertidas , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Maleabilidade , Dobramento de RNA , Repetições de TrinucleotídeosRESUMO
A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines (Ψ) and U1915 with 3-methyl-Ψ. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA and tested the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which were in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.
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Escherichia coli/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 23S/antagonistas & inibidores , Sequência de Bases , Conformação de Ácido Nucleico , Pseudouridina , RNA Bacteriano/antagonistas & inibidores , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismoRESUMO
RNAs perform multiple vital roles within cells, including catalyzing biological reactions and expression of proteins. Small RNA hairpins (sRNAh) are the smallest functional entities of nucleic acids and are involved in various important biological functions such as ligand binding and tertiary folding initiation of proteins. We investigated the conformational and free energy landscape of the sRNAh gcUUCGgc over a wide range of temperatures and pressures using fluorescence resonance energy transfer, Fourier-transform infrared and UV/Vis spectroscopy as well as small-angle X-ray scattering on the unlabeled and/or fluorescently labeled sRNAh. The sRNAh shows a broad melting profile with continuous increase of unpaired conformations up to about 60°C. However, the sRNAh structure might not be fully unfolded at temperatures as high as 90°C and still comprise various partially unfolded compact conformations. Pressure up to 400MPa has a small effect on the base pairing and base stacking interactions of the sRNAh, indicating small conformational perturbations, only, which might originate from minor changes in packing and hydration of the RNA molecule upon compression. Pressurization at 70°C, i.e. at a temperature above the melting transition, has no significant effect on the conformational ensemble of the sRNAh, i.e., it does not promote formation of new native stem connections after thermal denaturation. Finally, we noticed that Cy3/Cy5 labeling of the sRNAh changes, probably via stacking interactions between the fluorescent dyes and the nucleotide rings, the stability of the sRNAh, thereby rendering FRET analysis of the conformational dynamics of such small RNA structure inappropriate.