RESUMO
BACKGROUND: The aim of this study was to characterize the genomes of 30 Listeria monocytogenes isolates collected at a pig slaughterhouse to determine the molecular basis for their persistence. RESULTS: Comparison of the 30 L. monocytogenes genomes showed that successive isolates (i.e., persistent types) recovered from thew sampling site could be linked on the basis of single nucleotide variants confined to prophage regions. In addition, our study revealed the presence among these strains of the bcrABC cassette which is known to produce efflux pump-mediated benzalkonium chloride resistance, and which may account for the persistence of these isolates in the slaughterhouse environment. The presence of the bcrABC cassette was confirmed by WGS and PCR and the resistance phenotype was determined by measuring minimum inhibitory concentrations. Furthermore, the BC-resistant strains were found to produce lower amounts of biofilm in the presence of sublethal concentrations of BC. CONCLUSIONS: High resolution SNP-based typing and determination of the bcrABC cassette may provide a means of distinguishing between resident and sporadic L. monocytogenes isolates, and this in turn will support better management of this pathogen in the food industry.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Compostos de Benzalcônio/farmacologia , Farmacorresistência Bacteriana , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Matadouros/estatística & dados numéricos , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Genômica , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único , SuínosRESUMO
Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum.
Assuntos
Clostridium botulinum/genética , Flagelina/genética , Variação Genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , Canadá , Clostridium botulinum/classificação , Análise por Conglomerados , Genótipo , Ensaios de Triagem em Larga Escala , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analogue, we derived average interaction potentials (per square angstrom of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single-strand segments. These estimates, combined with the three interaction potentials and a term to account for interactions of urea with released ions, yield calculated m-values that are in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A-form conformation. The primary driving force for urea-induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though interactions of urea with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNAs are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with an increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states.
Assuntos
Desnaturação de Ácido Nucleico , RNA/química , Entropia , Conformação de Ácido Nucleico , RNA/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Termodinâmica , Ureia/químicaRESUMO
Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15-145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.
Assuntos
Vírus da Hepatite A , Norovirus , Rubus , Vírus da Hepatite A/genética , Norovirus/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ABSTRACT: A group of experts from all Canadian federal food safety partners was formed to monitor the potential issues relating to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) food contamination, to gather and consider all of the relevant evidence and to determine the impact for Canadian food safety. A comprehensive risk pathway was generated to consider the likelihood of a SARS-CoV-2 contamination event at any of the relevant steps of the food processing and handling chain and the potential for exposure and transmission of the virus to the consumer. The scientific evidence was reviewed and assessed for each event in the pathway, taking into consideration relevant elements that could increase or mitigate the risk of contamination. The advantage of having an event-wise contextualization of the SARS-CoV-2 transmission pathway through the food chain is that it provides a systematic and consistent approach to evaluate any new data and communicate its importance and impact. The pathway also increases the objectivity and consistency of the assessment in a rapidly evolving and high-stakes situation. Based on our review and analysis, there is currently no comprehensive epidemiological evidence of confirmed cases of SARS-CoV-2, or its known variants, causing coronavirus disease 2019 from transmission through food or food packaging. Considering the remote possibility of exposure through food, the likelihood of exposure by ingestion or contact with mucosa is considered negligible to very low, and good hygiene practices during food preparation should continue to be followed.
Assuntos
COVID-19 , SARS-CoV-2 , Canadá , Cadeia Alimentar , Humanos , HigieneRESUMO
Between 2016 and 2021, the Canadian Food Inspection Agency (CFIA) collected 4218 samples of fresh and frozen berries (blackberries, blueberries, raspberries, strawberries and mixed berries) and pomegranate arils at retail across 11 major cities in Canada and tested these samples for the presence of norovirus GI, norovirus GII and hepatitis A virus RNA. The purpose of this testing was to provide information on the prevalence of these viruses in berries and pomegranate arils on the Canadian marketplace. Of the 926 fresh fruit samples tested, norovirus GI RNA was detected in one raspberry sample and norovirus GII RNA was detected in one strawberry sample. Of the 3292 frozen fruit samples tested, norovirus GI RNA was detected in one blackberry sample, one raspberry sample and one strawberry sample, and norovirus GII RNA was detected in one blueberry sample, three raspberry samples, four strawberry samples, one pomegranate arils sample and one mixed berry sample. None of the fresh or frozen fruit samples tested positive for hepatitis A virus RNA. No statistically significant associations were observed between the prevalence of viral RNA in samples of fresh and frozen fruit, between the prevalence of viral RNA in samples of domestic and imported fruit or between the prevalence of viral RNA in samples of specific fruit types. Overall, the prevalence of norovirus GI and GII RNA together in fresh and frozen fruit samples in Canada was 0.36 %. The results of this study may be used to refine surveillance programs for norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils, e.g. by adapting the commodities tested and/or the numbers of planned samples to better target these hazards. This information may also be used to inform other Government of Canada approaches to better understand the controls associated norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils.
Assuntos
Mirtilos Azuis (Planta) , Fragaria , Vírus da Hepatite A , Norovirus , Punica granatum , Rubus , Canadá/epidemiologia , Microbiologia de Alimentos , Frutas , Vírus da Hepatite A/genética , Norovirus/genética , RNA Viral/genéticaRESUMO
Osmolytes are small organic molecules accumulated by cells in response to osmotic stress. Although their effects on protein stability have been studied, there has been no systematic documentation of their influence on RNA. Here, the effects of nine osmolytes on the secondary and tertiary structure stabilities of six RNA structures of differing complexity and stability have been surveyed. Using thermal melting analysis, m-values (change in DeltaG degrees of RNA folding per molal concentration of osmolyte) have been measured. All the osmolytes destabilize RNA secondary structure, although to different extents, probably because they favor solubilization of base surfaces. Osmolyte effects on tertiary structure, however, can be either stabilizing or destabilizing. We hypothesize that the stabilizing osmolytes have unfavorable interactions with the RNA backbone, which becomes less accessible to solvent in most tertiary structures. Finally, it was found that as a larger fraction of the negative charge of an RNA tertiary structure is neutralized by hydrated Mg(2+), the RNA becomes less responsive to stabilizing osmolytes and may even be destabilized. The natural selection of osmolytes as protective agents must have been influenced by their effects on the stabilities of functional RNA structures, though in general, the effects of osmolytes on RNA and protein stabilities do not parallel each other. Our results also suggest that some osmolytes can be useful tools for studying intrinsically unstable RNA folds and assessing the mechanisms of Mg(2+)-induced RNA stabilization.
Assuntos
Magnésio/química , Conformação de Ácido Nucleico , RNA/química , Sequência de Bases , Betaína/química , Cátions Bivalentes , Etilenoglicol/química , Metanol/química , Metilaminas/química , Dados de Sequência Molecular , Pressão Osmótica , Prolina/química , Estabilidade de RNA , Sacarose/química , Álcoois Açúcares/química , Termodinâmica , Ureia/químicaRESUMO
The application of new data streams generated from next-generation sequencing (NGS) has been demonstrated for food microbiology, pathogen identification, and illness outbreak detection. The establishment of best practices for data integrity, reproducibility, and traceability will ensure reliable, auditable, and transparent processes underlying food microbiology risk management decisions. We outline general principles to guide the use of NGS data in support of microbiological food safety. Regulatory authorities across intra- and international jurisdictions can leverage this effort to promote the reliability, consistency, and transparency of processes used in the derivation of genomic information for regulatory food safety purposes, and to facilitate interactions and the transfer of information in the interest of public health.
Assuntos
Inocuidade dos Alimentos , Genômica , Surtos de Doenças , Microbiologia de Alimentos , Reprodutibilidade dos TestesRESUMO
ITALIC! Salmonella entericasubspecies ITALIC! entericaserovar Berta has been isolated in multiple animal species and has been implicated in human disease. Here, we report a 4.7-Mbp draft genome sequence of ITALIC! S. entericaserovar Berta (ATCC strain 8392) and a nalidixic acid-resistant isolate derived from this strain.
RESUMO
Genomic antimicrobial resistance (AMR) prediction tools have the potential to support foodborne illness outbreak investigations through their application in the analysis of bacterial genomes from causative strains. The AMR marker profile of a strain of interest, initially identified in outbreak-associated clinical samples, may serve as the basis for customization of selective enrichment media, facilitating its recovery from samples in a food safety investigation. Different possibilities for AMR analyses include the use of comprehensive AMR gene databases such as the Comprehensive Antibiotic Resistance Database, which can be mined with in-house bioinformatics alignment tools (e.g., Antimicrobial Resistance Marker Identifier), or publicly available tools based on clinically relevant acquired AMR gene databases (e.g., ResFinder). In combination with a previously reported pipeline (SigSeekr) designed to identify specific DNA sequences associated with a particular strain for its rapid identification by PCR, it should be possible to deploy custom recovery and identification tools for the efficient detection of priority pathogens such as Shiga toxigenic Escherichia coli (STEC) outbreak strains within the time frame of an active investigation. Using a laboratory STEC strain as a model, trimethoprim resistance identified by both Antimicrobial Resistance Marker Identifier and ResFinder was used as the basis for its selective recovery against a background of commensal E. coli bacteria in ground beef samples. Enrichment in modified tryptic soy broth containing trimethoprim greatly enhanced the recovery of low numbers of model strain cells inoculated in ground beef samples, as verified by the enumeration of colonies on plating media using a strain-specific PCR method to determine the recovery efficiency for the target strain. We discuss the relative merits of different AMR marker prediction tools for this purpose and describe how such tools can be utilized to good effect in a typical outbreak investigation scenario.
Assuntos
Microbiologia de Alimentos , Escherichia coli Shiga Toxigênica/genética , Animais , Infecções por Escherichia coli/microbiologia , Doenças Transmitidas por Alimentos , Genômica , HumanosRESUMO
The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.
Assuntos
Infecções por Escherichia coli/diagnóstico , Toxina Shiga/genética , Proteínas de Escherichia coli/genética , Genômica , Humanos , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Shiga toxin-producing Escherichia coli strains, occasionally isolated from food, are of public health importance. Here, we report on the 5.30-Mbp draft genome sequence of E. coli O157:H7 EDL931 (strain ATCC 35150) and the 5.32-Mbp draft genome sequence of a nalidixic acid-resistant mutant derivative used as a distinguishable control strain in food-testing laboratories.
RESUMO
Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.
Assuntos
DNA Bacteriano/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Genoma Bacteriano , Reação em Cadeia da Polimerase/métodos , Biologia Computacional , Primers do DNA , DNA Bacteriano/genética , Escherichia coli O157/classificação , Escherichia coli O157/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Análise de Sequência de DNARESUMO
The timely identification and characterization of foodborne bacteria for risk assessment purposes is a key operation in outbreak investigations. Current methods require several days and/or provide low-resolution characterization. Here we describe a whole-genome-sequencing (WGS) approach (GeneSippr) enabling same-day identification of colony isolates recovered from investigative food samples. The identification of colonies of priority Shiga-toxigenic Escherichia coli (STEC) (i.e., serogroups O26, O45, O103, O111, O121, O145 and O157) served as a proof of concept. Genomic DNA was isolated from single colonies and sequencing was conducted on the Illumina MiSeq instrument with raw data sampling from the instrument following 4.5 hrs of sequencing. Modeling experiments indicated that datasets comprised of 21-nt reads representing approximately 4-fold coverage of the genome were sufficient to avoid significant gaps in sequence data. A novel bioinformatic pipeline was used to identify the presence of specific marker genes based on mapping of the short reads to reference sequence libraries, along with the detection of dispersed conserved genomic markers as a quality control metric to assure the validity of the analysis. STEC virulence markers were correctly identified in all isolates tested, and single colonies were identified within 9 hrs. This method has the potential to produce high-resolution characterization of STEC isolates, and whole-genome sequence data generated following the GeneSippr analysis could be used for isolate identification in place of lengthy biochemical characterization and typing methodologies. Significant advantages of this procedure include ease of adaptation to the detection of any gene marker of interest, as well as to the identification of other foodborne pathogens for which genomic markers have been defined.
Assuntos
Biologia Computacional/métodos , Genoma Bacteriano , Escherichia coli Shiga Toxigênica/genética , Mapeamento Cromossômico , Sondas de DNA/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/química , Microbiologia de Alimentos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNARESUMO
Salmonella enterica subsp. enterica serovar Mishmarhaemek is a Gram-negative, non-spore-forming, rod-shaped bacterium implicated in human clinical disease. Here, we report a 4.8-Mbp draft genome sequence of a nalidixic acid-resistant isolate of S. serovar Mishmarhaemek.
RESUMO
We have utilized the hairpin ribozyme, an RNA enzyme whose structure has been solved by high-resolution methods, to develop a new tool for mapping nucleobase-stacking interactions and potential metal-binding sites in RNA molecules. This tool involves the photoactivation of a specifically bound cobalt(III)hexaammine molecule at wavelengths corresponding to excitation of the metal ion complex only; no base excitation is involved. The photoexcitation initiates a process which strongly promotes the formation of a novel covalent bond or crosslink between one base (termed the "first base"), which is close in space to the excited cobalt(III)hexaammine complex, and another base upon which the first base is closely stacked. These crosslinked species can be isolated and sequenced; their activities can be analyzed to ensure that the crosslinked structures represent an active conformation of the molecule. We have shown that, as in electron transfer in DNA, several criteria must be met to result in the successful formation of these crosslinks. These include the appropriate oxidation potential of the first donor base, the stacking and close interaction of the two donor bases involved in the crosslink, and the binding of a specific cobalt(III)hexaammine molecule to the first donor base. Additionally, we have determined that this crosslinking is pH-sensitive, although the cause of this sensitivity remains unknown. This tool has proven useful in the past for the analysis of the hairpin ribozyme folded structure, and has been applied to identify potential metal-binding sites on the hairpin and extended hammerhead ribozymes.
Assuntos
Aminas/química , Cobalto/química , Reagentes de Ligações Cruzadas/química , Compostos Organometálicos/química , RNA Catalítico/química , Concentração de Íons de Hidrogênio , Modelos Químicos , Conformação de Ácido NucleicoRESUMO
The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are â¼0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol(-1)m(-1) for four RNA tertiary structures (30-86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg(2+) and that the structures stabilized by TMAO and Mg(2+) are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg(2+) ion chelation site formed in part by a buried phosphate, even though Mg(2+) is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, +211 cal mol(-1)m(-1) for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66-173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg(2+) suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.
Assuntos
Magnésio/metabolismo , Metilaminas/metabolismo , Conformação de Ácido Nucleico , Fosfatos/metabolismo , RNA/química , RNA/metabolismo , Radical Hidroxila/metabolismo , Modelos Moleculares , Espalhamento a Baixo Ângulo , Água/metabolismoRESUMO
RNA secondary and tertiary structures are strongly stabilized by added salts, and a quantitative thermodynamic analysis of the relevant ion-RNA interactions is an important aspect of the RNA folding problem. Because of long-range electrostatic forces, an RNA perturbs the distribution of both cations and anions throughout a large volume. Binding formalisms that require a distinction between "bound" and "free" ions become problematic in such situations. A more fundamental thermodynamic framework is developed here, based on preferential interaction coefficients; linkage equations derived from this framework provide a model-free description of the "uptake" or "release" of cations and anions that accompany an RNA conformational transition. Formulas appropriate for analyzing the dependence of RNA stability on either mono- or divalent salt concentration are presented and their application to experimental data is illustrated. Two example datasets are analyzed with respect to the monovalent salt dependence of tertiary structure formation in different RNAs, and three different experimental methods for quantitating the "uptake" of Mg(2+) ions are applied to the folding of a riboswitch RNA. Advantages and limitations of each method are discussed.
Assuntos
Íons/química , Modelos Teóricos , RNA/química , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
Many RNA tertiary structures are stable in the presence of monovalent ions alone. To evaluate the degree to which ions at or near the surfaces of such RNAs contribute to stability, the salt-dependent stability of a variety of RNA structures was measured with each of the five group I cations. The stability of hairpin secondary structures and a pseudoknot tertiary structure are insensitive to the ion identity, but the tertiary structures of two other RNAs, an adenine riboswitch and a kissing loop complex, become more stable by 2-3 kcal/mol as ion size decreases. This "default" trend is attributed to the ability of smaller ions to approach the RNA surface more closely. The degree of cation accumulation around the kissing loop complex was also inversely proportional to ion radius, perhaps because of the presence of sterically restricted pockets that can be accessed only by smaller ions. An RNA containing the tetraloop-receptor motif shows a strong (up to approximately 3 kcal/mol) preference for Na(+) or K(+) over other group I ions, consistent with the chelation of K(+) by this motif in some crystal structures. This RNA reverts to the default dependence on ion size when a base forming part of the chelation site is mutated. Lastly, an RNA aptamer for cobinamide, which was originally selected in the presence of high concentrations of LiCl, binds ligand more strongly in the presence of Li(+) than other monovalent ions. On the basis of these trends in RNA stability with group I ion size, it is argued that two features of RNA tertiary structures may promote strong interactions with ions at or near the RNA surface: negative charge densities that are higher than that in secondary structures, and the occasional presence of chelation sites, which are electronegative pockets that selectively bind ions of an optimum size.
Assuntos
Metais Alcalinos/química , Conformação de Ácido Nucleico , RNA/química , Amônia/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Cátions Monovalentes/química , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade de RNA , Termodinâmica , Vitamina B 12/químicaRESUMO
Metal ions facilitate the folding of the hairpin ribozyme but do not participate directly in catalysis. The metal complex cobalt(III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by [Co(NH3)6]3+ is different from that organized by Mg2+ and that revealed in the crystal structure [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786] (1). Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in [Co(NH3)6]3+. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70-fold showed only subtle decreases or even increases in observed rates when assayed in [Co(NH3)6]3+. Here, we propose a model of the [Co(NH3)6]3+-mediated catalytic core generated by MC-SYM that is consistent with these data.