Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain
Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain
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DOI：
10.1021/bi011570u
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发表时间：
2001-12-11
期刊：
影响因子：
2.9
通讯作者：
Nicholson, AW
中科院分区：
文献类型：
--
作者：
Sun, WM;Jun, E;Nicholson, AW
核糖核酸酶III超家族代表了一组结构相关的双链（ds）特异性内切核糖核酸酶，其在多种原核和真核RNA成熟和降解途径中发挥关键作用。dsRNA结合结构域（dsRBD）是超家族的保守特征，并且对于底物识别是重要的。RNase III家族成员也表现出“催化”结构域，部分由一组高度保守的氨基酸定义，其中至少一个（谷氨酸）对切割重要，但对底物结合不重要。然而，尚不清楚催化结构域是否需要dsRBD来获得活性。本报告表明，缺乏dsRBD的大肠杆菌RNase III的截短形式（RNase III[Delta dsRBD]）可以在体外准确切割小的加工底物。RNase III[Delta dsRBD]的最佳活性在低盐浓度（25 mM）或Mn 2+（类似于5 mM）下观察到。在60 mM Na+和5 mM Mn 2+下，RNase III[Delta dsRBD]的催化效率与RNase III在生理盐浓度和Mg 2+下的催化效率相似。在Mg 2+存在下，RNase III[Delta dsRBD]的效率低于野生型酶，因为K-m较高。与RNase III类似，RNase III[Delta dsRBD]被高浓度的Mn 2+抑制，这是由于金属离子占据了酶上的抑制位点。RNase III[Delta dsRBD]保留了对双链RNA的严格特异性，这表明它无法切割（rA）（25）、（rU）（25）或（rC）（25）。此外，dsDNA、ssDNA或RNA-DNA杂合体不被切割。低（微摩尔）浓度的溴化乙锭阻断RNA酶III[Delta dsRBD]对底物的切割，这与RNA酶III的抑制作用相似，表明抑制作用为嵌入模式。最后，RNase III[Delta dsRBD]对特异性沃森-克里克碱基对取代敏感，这也抑制RNase III。这些发现支持RNase III的作用机制，其中催化结构域（i）可以独立于dsRBD发挥作用，（ii）是dsRNA特异性的，（iii）参与切割位点选择。
The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[Delta dsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[Delta dsRBD] is observed at low salt concentrations (25 mM) or Mn2+ (similar to5 mM). At 60 mM Na+ and 5 mM Mn2+ the catalytic efficiency of RNase III[Delta dsRBD] is similar to that of RNase III at physiological salt concentrations and Mg2+. In the presence of Mg2+ RNase III[Delta dsRBD] is less efficient than the wild-type enzyme, due to a higher K-m. Similar to RNase III, RNase III[Delta dsRBD] is inhibited by high concentrations of Mn2+, which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[Delta dsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[Delta dsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[Delta dsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.