New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2021 May 21;49(9):5265-5277. doi: 10.1093/nar/gkab284.
Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.
自从最初对其进行描述以来,大肠杆菌 RNase I 一直被描述为一种单链特异性 RNA 内切酶,它以很大程度上序列非依赖的方式切割其底物。在这里,我们描述了 RNase I 在双链 RNA(dsRNA)上的强钙(Ca2+)依赖性活性,以及一种 Ca2+依赖性新型杂交酶活性,可在 DNA:RNA 杂交体中消化 RNA 链。令人惊讶的是,Ca2+ 不会影响 RNase I 对单链 RNA(ssRNA)的活性,这表明 Ca2+ 在调节 RNase I 活性方面具有特定作用。RNase I 上一个以前被忽视的 Ca2+ 结合位点的突变导致酶获得功能,使其在 dsRNA 上高度活跃,并且不再被金属刺激。总之,我们的数据表明,天然 RNase I 含有结合的 Ca2+,使其能够靶向单链和双链 RNA,从而具有比最初为这种传统酶提出的更广泛的底物特异性。此外,dsRNase 活性而不是 ssRNase 活性与 RNase I 的 Ca2+依赖性相关,这一发现可能作为应用分子生物学中的工具很有用。