RESUMO
Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Here, we characterized the sequence specificity of eight other members of the Mini-III RNase family from different bacterial species. High-throughput analysis of the cleavage products of Φ6 bacteriophage dsRNA indicated subtle differences in sequence preference between these RNases, which were confirmed and characterized by systematic analysis of the cleavage kinetics of a set of short dsRNA substrates. We also showed that the sequence specificities of Mini-III RNases are not reflected by different binding affinities for cognate and non-cognate sequences, suggesting that target selection occurs predominantly at the cleavage step. We were able to identify two structural elements, the α4 helix and α5b-α6 loop that were involved in target selection. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research.
Assuntos
Elementos Estruturais de Proteínas , Ribonuclease III/química , Sequência de Aminoácidos , Bacteriófagos/enzimologia , Bacteriófagos/genética , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Motivos de Nucleotídeos , Clivagem do RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Ribonuclease III/química , Ribonuclease III/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
This pilot study presents preliminary results on interrelations between alkali and alkaline earth elements during their transfer to mycelium and fruitbodies of saprophytic fungi. The accumulation and distribution of four elements (cesium, potassium, sodium, and calcium) was evaluated in king oyster mushroom (Pleurotus eryngii) cultivated under controlled conditions. Elemental composition of caps, stipes, and the substrate was analyzed by atomic absorption/emission spectroscopy to evaluate discrimination, concentration, and transfer factors. The transfer factors determined for all the investigated elements were different and can be put in the following order: Cs > K > Na > Ca. There has been a higher accumulation of cesium in caps than in stipes. Distribution of cesium in fruitbodies depended on the presence of other ions in the substrate. The addition of Ca2+ limited the transport of cesium and potassium from stipes to caps. Sodium and calcium were mainly accumulated in the stipes. In a control experiment, without supplementation with K+, Na+, and Ca2+, approximately 62% of the cesium present in the substrate was extracted by mycelium and transported to the fruitbodies. Possible applications of fruiting saprophytic fungi in bioremediation are discussed.