RESUMEN
The structures of four small RNAs each containing a different version of the UNAC loop were determined in solution using NMR spectroscopy and restrained molecular dynamics. The UMAC tetraloops (where M is A or C) exhibited a typical GNRA fold including at least one hydrogen bond between the first U and fourth C. In contrast, UGAC and UUAC tetraloops have a different orientation of the first and fourth residues, such that they do not closely mimic the GNRA fold. Although the UMAC tetraloops are excellent structural mimics of the GNRA tetraloop backbone, sequence comparisons typically do not reveal co-variation between the two loop types. The limited covariation is attributed to differences in the location of potential hydrogen bond donors and acceptors as a result of the replacement of the terminal A of GNRA with C in the UMAC version. Thus, UMAC loops do not readily form the common GNRA tetraloop-receptor interaction. The loop at positions 863-866 in E. coli 16S ribosomal RNA appears to be a major exception. However, in this case the GNRA loop does not in fact engage in the usual base to backbone tertiary interactions. In summary, UMAC loops are not just an alternative sequence version of the GNRA loop family, but instead they expand the types of interactions, or lack thereof, that are possible. From a synthetic biology perspective their inclusion in an artificial RNA may allow the establishment of a stable loop structure while minimizing unwanted long range interactions or permitting alternative long-range interactions. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 617-628, 2012.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , Pliegue del ARN , ARN Ribosómico/química , Bacterias , Escherichia coli , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , ARN Ribosómico/síntesis químicaRESUMEN
Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5'cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.
Asunto(s)
Humanos , Animales , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Eucariotas/citología , Células Eucariotas/fisiología , Células Eucariotas/virología , Factores Eucarióticos de Iniciación/análisis , Factores Eucarióticos de Iniciación/biosíntesis , Factores Eucarióticos de Iniciación/genética , Proteínas , ARN Ribosómico/análisis , ARN Ribosómico/biosíntesis , ARN Ribosómico/síntesis químicaRESUMEN
Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific. Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides. In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series. Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding. The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes. Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2. However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine. In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria. L-Neamine inhibits the growth of E. coli and P. aeruginosa with 8- and 3-fold higher MIC than D-neamine. Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E. coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series. These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.
Asunto(s)
Antibacterianos/química , ARN Ribosómico/química , Antibacterianos/farmacología , Unión Competitiva , Polarización de Fluorescencia/métodos , Framicetina/química , Framicetina/farmacología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Oligorribonucleótidos/síntesis química , Paromomicina/química , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bacteriano/antagonistas & inhibidores , ARN Bacteriano/síntesis química , ARN Bacteriano/química , ARN de Hongos/antagonistas & inhibidores , ARN de Hongos/síntesis química , ARN de Hongos/química , ARN Ribosómico/antagonistas & inhibidores , ARN Ribosómico/síntesis química , Rodaminas/metabolismo , Ribosomas/química , Ribosomas/efectos de los fármacos , Espectrometría de Fluorescencia , Estereoisomerismo , Tobramicina/químicaRESUMEN
The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.
Asunto(s)
Aminoglicósidos/química , Antibacterianos/farmacología , Kanamicina/análogos & derivados , ARN Ribosómico/metabolismo , Algoritmos , Aminoglicósidos/metabolismo , Bacterias , Polarización de Fluorescencia , Framicetina/química , Framicetina/farmacología , Humanos , Kanamicina/química , Kanamicina/farmacología , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Paromomicina/química , Paromomicina/farmacología , ARN Ribosómico/síntesis química , ARN Ribosómico/química , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 18S/química , ARN Ribosómico 18S/metabolismo , Rodaminas/química , Rodaminas/farmacología , Relación Estructura-Actividad , Tobramicina/química , Tobramicina/farmacologíaRESUMEN
Hairpins are the most common elements of RNA secondary structure, playing important roles in RNA tertiary architecture and forming protein binding sites. Triloops are common in a variety of naturally occurring RNA hairpins, but little is known about their thermodynamic stability. Reported here are the sequences and thermodynamic parameters for a variety of stable and unstable triloop hairpins. Temperature gradient gel electrophoresis (TGGE) can be used to separate a simple RNA combinatorial library based on thermal stability [Bevilacqua, J. M., and Bevilacqua, P. C. (1998) Biochemistry 45, 15877-15884]. Here we introduce the application of TGGE to separating and analyzing a complex RNA combinatorial library based on thermal stability, using an RNA triloop library. Several rounds of in vitro selection of an RNA triloop library were carried out using TGGE, and preferences for exceptionally stable and unstable closing base pairs and loop sequences were identified. For stable hairpins, the most common closing base pair is CG, and U-rich loop sequences are preferred. Closing base pairs of GC and UA result in moderately stable hairpins when combined with a stable loop sequence. For unstable hairpins, the most common closing base pairs are AU and UG, and U-rich loop sequences are no longer preferred. In general, the contributions of the closing base pair and loop sequence to overall hairpin stability appear to be additive. Thermodynamic parameters for individual hairpins determined by UV melting are generally consistent with outcomes from selection experiments, with hairpins containing a CG closing base pair having a DeltaDeltaG degrees (37) 2.1-2.5 kcal/mol more favorable than hairpins with other closing base pairs. Sequences and thermodynamic rules for triloop hairpins should aid in RNA structure prediction and determination of whether naturally occurring triloop hairpins are thermodynamically stable.
Asunto(s)
Conformación de Ácido Nucleico , ARN Ribosómico/química , ARN Ribosómico/aislamiento & purificación , Composición de Base , Clonación Molecular , Técnicas Químicas Combinatorias , Electroforesis en Gel de Poliacrilamida , Biblioteca de Genes , Calor , ARN Ribosómico/síntesis química , ARN Ribosómico/metabolismo , Termodinámica , Rayos UltravioletaRESUMEN
Nine different hammerhead RNA self-cleaving domains consistent with the consensus secondary structure proposed by Keese and Symons (1987) were prepared and tested for cleavage. Each hammerhead was constructed from two oligoribonucleotides in two different configurations. Although cleavage was observed in all nine cases, the rates of cleavage varied by more than a thousand fold. The presence of RNA secondary structure incompatible with hammerhead formation in the individual oligos may be responsible for the large rate differences. We have also examined the degree of participation of a proposed dimer hammerhead intermediate in one case and conclude that, while such a four-stranded structure can form, it is not the preferred reaction intermediate.
Asunto(s)
Plantas/genética , Empalme del ARN , ARN Ribosómico/genética , Secuencia de Bases , Catálisis , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , ARN Catalítico , ARN Ribosómico/síntesis química , Relación Estructura-ActividadRESUMEN
RNA enzymes (ribozymes) which can cleave RNA by recognizing sequences of 9-15 bases are described. Substrates must contain UX (X = U, C or A). A ribozyme consisting of two oligoribonucleotides (19 mer and 15 mer) was shown to cleave a ribo 11 mer catalytically with Km and kcat values of 0.53 microM and 0.03 min-1, respectively. A non-cleavable substrate-ribozyme complex containing 2'-O-methylnucleoside was prepared and CD spectra were compared at different temperature. In order to obtain an efficient ribozyme, a one-strand RNA with a chain length of 37 was prepared. The ribozyme was shown to distinguish a single base mutation in mRNA's which were prepared by transcription of two synthetic DNA duplexes coding for positions 7-26 of c-Ha-ras protein. The mutant (Val-12) mRNA which had GUU was cleaved but the wild type mRNA which contained GGU was not changed, when treated by the ribozymes in the presence of Mg2+.
Asunto(s)
ARN Ribosómico/metabolismo , ARN/metabolismo , Secuencia de Bases , Cinética , Magnesio/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , ARN/genética , Empalme del ARN , ARN Catalítico , ARN Ribosómico/síntesis química , Especificidad por SustratoRESUMEN
The chloroethoxyethyl (CEE) group is completely stable under the acidic conditions required to remove the 5'-protecting groups in the oligoribonucleotide synthesis, but can be cleaved under the similar condition to that of the tetrahydropyranyl (THP) group in region of pH 2-3. The oligoribonucleotides were synthesized by the phosphoramidite method on solid supports.
Asunto(s)
Oligorribonucleótidos/síntesis química , ARN Ribosómico/síntesis química , Tetrahymena/genética , Animales , Uridina/análogos & derivados , Uridina/síntesis químicaRESUMEN
Fifteen RNA hairpins that share the same stem sequence and have homopolymer loops of A, C and U residues which vary in length from three to nine nucleotides were synthesized and their thermal stabilities determined. Tm varies as a function of loop size but is almost independent of loop composition. Loops of four or five nucleotides are found to be the most stable loop size. This is consistent with the observation that four-membered loops are the most prevalent loop size in 16S-like RNAs. The contribution of each loop to hairpin stability was calculated by subtracting the known contribution of the helical stem. These data should be useful for predicting the stability of other hairpins.
Asunto(s)
Conformación de Ácido Nucleico , ARN Ribosómico , Composición de Base , Secuencia de Bases , Desnaturalización de Ácido Nucleico , Filogenia , ARN Ribosómico/síntesis química , ARN Ribosómico/aislamiento & purificación , Moldes Genéticos , Termodinámica , Transcripción GenéticaRESUMEN
The self-excision of a 413-base intervening sequence of the 26S rRNA of Tetrahymena thermophila has been investigated using phosphorothioate-substituted RNA. Transcripts containing this intron were prepared by T7 RNA polymerase-catalyzed polymerisation using a M13 mICE10 vector in the presence of various nucleoside alpha-thiotriphosphate analogues. Wild-type transcripts incorporating phosphorothioates 5' to adenosine or uridine were inactive, whereas incorporation 5' to cytidine or guanosine allowed splicing. The first two substitutions place phosphorothioates inter alia at the 5' and 3' splice sites respectively. Mutagenesis at either site allowed phosphorothioate substitution 5' to guanosine at each splice site. This did not block splicing, suggesting that substitution at internal sites within the intron has more effect.
Asunto(s)
Empalme del ARN , ARN Ribosómico/genética , Tetrahymena/genética , Animales , Secuencia de Bases , Exones , Indicadores y Reactivos , ARN Ribosómico/síntesis química , Tionucleótidos , Transcripción GenéticaRESUMEN
A ribosome, the cellular site for protein synthesis, is a very complex organelle composed of a myriad of macromolecular substructures. As models for this complex structure, we have been examining the structures and interactions of eukaryotic 5S and 5.8S rRNAs using adaptations of rapid RNA gel sequencing techniques. Estimates for their higher order structures have been proposed or evaluated, sites of interaction with other ribosomal components have been delineated, and the topography of these RNAs within the intact ribosome or 60S subunit have been examined. The results indicate that a universal structure for the ribosomal RNAs may only be present within the ribosome, that these molecules are probably present, at least in part, within the ribosomal interface, and that the bases for interactions with other ribosomal components are strongly dependent on their higher order structure. The experimental approaches which underlie these studies are considered in this review and the significance of the results with respect to the function and evolution of the ribosome are briefly discussed.