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Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases

机译：鉴定负责Mini-III RNase之间序列偏好多样性的蛋白质结构元件

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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.
机译：许多已知的核糖核酸内切酶基于在切割位点处或附近存在一个或几个特定核苷酸来选择其底物。在某些情况下，选择性还取决于基底的结构特征。我们最近描述了来自枯草芽孢杆菌的Mini-III RNase对双链RNA的序列特异性切割。在这里，我们表征了来自不同细菌物种的Mini-III RNase家族的其他八个成员的序列特异性。 Φ6噬菌体dsRNA裂解产物的高通量分析表明，这些RNase之间的序列偏好之间存在细微差异，这已通过系统分析一组短dsRNA底物的裂解动力学得到了证实和表征。我们还表明，Mini-III RNase的序列特异性不能通过同源和非同源序列的不同结合亲和力反映出来，这表明靶标选择主要发生在裂解步骤。我们能够确定参与目标选择的两个结构元素，即α4螺旋和α5b-α6环。表征八种Mini-III RNase的序列特异性可为更好地了解Mini-III RNase识别RNA底物并将这些酶及其工程衍生物用作RNA研究工具提供基础。

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