RESUMO
In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.
Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA , Regulação Viral da Expressão Gênica , Sequências Hélice-Volta-Hélice , Proteínas de Ligação a RNA , Proteínas Virais , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Sítios de Ligação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Genes Virais , Modelos Moleculares , Conformação de Ácido Nucleico , Pectobacterium carotovorum/virologia , Biossíntese de Proteínas/genética , Domínios Proteicos , Ribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Especificidade por Substrato , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestruturaRESUMO
Alternative splicing (AS) is a complex process that generates transcript variants from a single pre-mRNA and is involved in numerous biological functions. Many RNA-binding proteins are known to regulate AS; however, little is known about the underlying mechanisms, especially outside the mammalian clade. Here, we show that polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana regulate AS of cassette exons via pyrimidine (Py)-rich motifs close to the alternative splice sites. Mutational studies on three PTB-dependent cassette exon events revealed that only some of the Py motifs in this region are critical for AS. Moreover, in vitro binding of PTBs did not reflect a motif's impact on AS in vivo. Our mutational studies and bioinformatic investigation of all known PTB-regulated cassette exons from A. thaliana and human suggested that the binding position of PTBs relative to a cassette exon defines whether its inclusion or skipping is induced. Accordingly, exon skipping is associated with a higher frequency of Py stretches within the cassette exon, and in human also upstream of it, whereas exon inclusion is characterized by increased Py motif occurrence downstream of said exon. Enrichment of Py motifs downstream of PTB-activated 5' splice sites is also seen for PTB-dependent intron removal and alternative 5' splice site events from A. thaliana, suggesting this is a common step of exon definition. In conclusion, the position-dependent AS regulatory mechanism by PTB homologs has been conserved during the separate evolution of plants and mammals, while other critical features, in particular intron length, have considerably changed.
Assuntos
Processamento Alternativo , Proteínas de Arabidopsis , Arabidopsis , Éxons , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Arabidopsis/genética , Arabidopsis/metabolismo , Éxons/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pirimidinas , HumanosRESUMO
Research in the last two decades has increasingly demonstrated that RNA has capabilities comparable to those of proteins, for example the ability to form intricate 3D structures necessary for catalysis. Numerous protein domains are known in varied within-domain rearrangements, called permutations, that change the N- to C-terminal order of important amino acids inside the domain, but maintain their 3D locations. In RNAs, by contrast, only simple circular permutations are known, in which 5' and 3' portions of the molecule are merely swapped. Here, we computationally find and experimentally validate naturally occurring RNAs exhibiting non-circular permutations of previously established hammerhead ribozyme RNAs. In addition to the rearranged RNAs, a bioinformatics-based search uncovered many other new conserved RNA structures that likely play different biological roles. Our results further demonstrate the structural sophistication of RNA, indicate a need for more nuance in the analysis of pseudoknots, and could be exploited in RNA-based biotechnology applications.
Assuntos
RNA Catalítico , RNA Catalítico/química , RNA Catalítico/metabolismo , Conformação de Ácido Nucleico , CinéticaRESUMO
Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.
Assuntos
Riboswitch , Guanidina/química , Guanidina/metabolismo , Conformação de Ácido Nucleico , Ligantes , Guanidinas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismoRESUMO
Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory ("metalloregulatory") proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni(2+) and Co(2+). These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co(2+)-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobalto/metabolismo , Níquel/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Riboswitch/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte de Cátions/genética , Clostridium/genética , Clostridium/metabolismo , Sequência Conservada , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Self-cleaving ribozymes are catalytic RNAs that cut themselves at a specific inter-nucleotide linkage. They serve as a model of RNA catalysis, and as an important tool in biotechnology. For most of the nine known structural classes of self-cleaving ribozymes, at least hundreds of examples are known, and some are present in multiple domains of life. By contrast, only four unique examples of the hairpin ribozyme class are known, despite its discovery in 1986. We bioinformatically predicted 941 unique hairpin ribozymes of a different permuted form from the four previously known hairpin ribozymes, and experimentally confirmed several diverse predictions. These results profoundly expand the number of natural hairpin ribozymes, enabling biochemical analysis based on natural sequences, and suggest that a distinct permuted form is more biologically relevant. Moreover, all novel hairpins were discovered in metatranscriptomes. They apparently reside in RNA molecules that vary both in size-from 381 to 5170 nucleotides-and in protein content. The RNA molecules likely replicate as circular single-stranded RNAs, and potentially provide a dramatic increase in diversity of such RNAs. Moreover, these organisms have eluded previous attempts to isolate RNA viruses from metatranscriptomes-suggesting a significant untapped universe of viruses or other organisms hidden within metatranscriptome sequences.
Assuntos
RNA Catalítico/química , RNA Circular/química , Biologia Computacional , Conformação de Ácido Nucleico , RNA Catalítico/metabolismoRESUMO
Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org.
Assuntos
Bases de Dados de Ácidos Nucleicos , Metagenoma , MicroRNAs/genética , RNA Bacteriano/genética , RNA não Traduzido/genética , RNA Viral/genética , Bactérias/genética , Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Humanos , Internet , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/classificação , RNA Bacteriano/metabolismo , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , RNA Viral/classificação , RNA Viral/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA , Software , Vírus/genética , Vírus/metabolismoRESUMO
Guanidine is sensed by at least four different classes of riboswitches that are widespread in bacteria. However, only very few insights into physiological roles of guanidine exist. Genes predominantly regulated by guanidine riboswitches are Gdx transporters exporting the compound from the bacterial cell. In addition, urea/guanidine carboxylases and associated hydrolases and ABC transporters are often found combined in guanidine-inducible operons. We noted that the associated ABC transporters are configured to function as importers, challenging the current view that riboswitches solely control the detoxification of guanidine in bacteria. We demonstrate that the carboxylase pathway enables utilization of guanidine as sole nitrogen source. We isolated three enterobacteria (Raoultella terrigena, Klebsiella michiganensis, and Erwinia rhapontici) that utilize guanidine efficiently as N-source. Proteome analyses show that the expression of a carboxylase, associated hydrolases and transport genes is strongly induced by guanidine. Finding two urea/guanidine carboxylase enzymes in E. rhapontici, we demonstrate that the riboswitch-controlled carboxylase displays specificity toward guanidine, whereas the other enzyme prefers urea. We characterize the distribution of riboswitch-associated carboxylases and Gdx exporters in bacterial habitats by analyzing available metagenome data. The findings represent a paradigm shift from riboswitch-controlled detoxification of guanidine to the uptake and assimilation of this enigmatic nitrogen-rich compound.
Assuntos
Enterobacteriaceae/metabolismo , Erwinia/metabolismo , Guanidina/metabolismo , Klebsiella/metabolismo , Riboswitch/genética , Carbono-Nitrogênio Ligases/genética , Metabolismo Energético/genética , Regulação Bacteriana da Expressão Gênica/genética , Hidrolases/metabolismo , Proteínas de Membrana Transportadoras/genéticaRESUMO
Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , RNA Bacteriano/genética , RNA/genética , Riboswitch/genética , Biologia Computacional , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Guanidina/metabolismo , Proteínas de Membrana Transportadoras/genética , Conformação de Ácido Nucleico , Domínios Proteicos/genética , Staphylococcus aureus/genéticaRESUMO
Small endonucleolytic ribozymes promote the self-cleavage of their own phosphodiester backbone at a specific linkage. The structures of and the reactions catalysed by members of individual families have been studied in great detail in the past decades. In recent years, bioinformatics studies have uncovered a considerable number of new examples of known catalytic RNA motifs. Importantly, entirely novel ribozyme classes were also discovered, for most of which both structural and biochemical information became rapidly available. However, for the majority of the new ribozymes, which are found in the genomes of a variety of species, a biological function remains elusive. Here, we concentrate on the different approaches to find catalytic RNA motifs in sequence databases. We summarize the emerging principles of RNA catalysis as observed for small endonucleolytic ribozymes. Finally, we address the biological functions of those ribozymes, where relevant information is available and common themes on their cellular activities are emerging. We conclude by speculating on the possibility that the identification and characterization of proteins that we hypothesize to be endogenously associated with catalytic RNA might help in answering the ever-present question of the biological function of the growing number of genomically encoded, small endonucleolytic ribozymes.
Assuntos
Biologia Computacional/métodos , Motivos de Nucleotídeos/genética , RNA Catalítico/genética , Análise de Sequência de RNA/métodos , Catálise , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/isolamento & purificaçãoRESUMO
S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.
Assuntos
Biossíntese de Proteínas , Riboswitch/genética , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Evolução Molecular , Genômica , Ligantes , RNA/química , RNA/genética , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
BACKGROUND: RNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5' UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted. RESULTS: By analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites. CONCLUSIONS: This work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.
Assuntos
Regiões 5' não Traduzidas , Archaea/genética , Bactérias/genética , RNA Ribossômico/química , Archaea/metabolismo , Bacillus subtilis/genética , Bactérias/metabolismo , Escherichia coli/genética , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/metabolismoRESUMO
Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2'-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.
Assuntos
Biologia Computacional/métodos , RNA/química , RNA/genética , Riboswitch/genética , Sequência de Bases , Sítios de Ligação , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Desoxiguanosina/química , Glicina/química , Guanina/química , Ligantes , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.
Assuntos
RNA Bacteriano/química , RNA não Traduzido/química , Sequências Reguladoras de Ácido Ribonucleico , Integrons , Motivos de Nucleotídeos , Plasmídeos/genética , Transcrição ReversaRESUMO
Five distinct riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA structures that can form to selectively recognize SAM, and showcase the ability of RNA to utilize diverse strategies to perform similar biological functions.
Assuntos
Bifidobacterium/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , S-Adenosilmetionina/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Bifidobacterium/química , Bifidobacterium/metabolismo , Sítios de Ligação , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RiboswitchRESUMO
Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evidenced by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein we exploited this association to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. We identified three new self-cleaving classes, which we named twister sister, pistol and hatchet, from this collection, suggesting that even more ribozymes remain hidden in modern cells.
Assuntos
Archaea/genética , Bactérias/genética , Genômica/métodos , RNA Catalítico/química , Algoritmos , Archaea/enzimologia , Bactérias/enzimologia , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Biossíntese de Proteínas , Proteólise , RNA Catalítico/genética , RNA Catalítico/metabolismoRESUMO
Hammerhead ribozymes represent the most common of the 9 natural classes of self-cleaving RNAs. The hammerhead catalytic core includes 11 highly-conserved nucleotides located largely within the unpaired regions of a junction formed by stems I, II and III. The vast majority of previously reported examples carry an additional pseudoknot or other tertiary interactions between nucleotides that precede stem I and nucleotides in the loop of stem II. These extra contacts are critical for high-speed RNA catalysis. Herein, we report the discovery of â¼150,000 additional variant hammerhead representatives that exhibit diminished stem III substructures. These variants are frequently associated with Penelope-like retrotransposons, which are a type of mobile genetic element. Kinetic analyses indicate that these RNAs form dimers to cleave RNA.
Assuntos
Clivagem do RNA , RNA Catalítico/química , RNA/metabolismo , Retroelementos , Animais , Pareamento de Bases , Sequência de Bases , Biocatálise , Domínio Catalítico , Dimerização , Isópteros/química , Cinética , Conformação de Ácido Nucleico , RNA/química , RNA/genética , RNA Catalítico/genética , RNA Catalítico/isolamento & purificação , RNA Catalítico/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Solo/química , Urocordados/químicaRESUMO
Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2,700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.
Assuntos
Biologia Computacional , RNA Catalítico/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/química , Estrutura Secundária de ProteínaRESUMO
Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Notably, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with subnanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.
Assuntos
Bacillus subtilis/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Riboswitch/fisiologia , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Conformação de Ácido Nucleico , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de Sinais , LevedurasRESUMO
Estimates of the total number of bacterial species indicate that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins and RNAs. Bioinformatics searches of genomic DNA from bacteria commonly identify new noncoding RNAs (ncRNAs) such as riboswitches. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, indicating that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.