In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the C-terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic N-terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.

中文翻译：

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衔接蛋白 RapZ 通过蛋白-蛋白相互作用激活核糖核酸内切酶 RNase E 以切割小调节 RNA

在大肠杆菌中，内切核糖核酸酶 RNase E 启动许多 RNA 的降解，并代表转录后调控的枢纽。四聚体衔接蛋白 RapZ 将小调节 RNA GlmZ 靶向至被 RNase E 降解。 RapZ 通过位于 C 端的结构域结合 GlmZ 并与 RNase E 相互作用，促进碱基配对区域中的 GlmZ 裂解。必要时，GlmZ 的裂解会被同源小 RNA GlmY 抵消，GlmY 通过分子模拟隔离 RapZ。在当前的研究中，我们解决了 RapZ 采用的分子机制。我们表明，当以增加的浓度提供时，RapZ 突变体在 RNA 结合方面受损但精通结合 RNase E，能够在体内和体外刺激 GlmZ 裂解。相比之下，截断的 RapZ 变体保留了 RNA 结合活性，但无法接触 RNase E，因此缺乏这种活性。一致地，我们发现四聚体 RapZ 通过与催化 N 末端内的大球状结构域直接相互作用而结合同样的四聚体 RNase E，独立于 RNA。尽管 RapZ 在体外刺激至少一种非同源 RNA 被 RNase E 切割，但如 RNA 测序所揭示的，其在体内的活性仅限于 GlmZ，这表明切割也需要 RNA 底物中的某些特征。总之，RapZ 通过与其催化结构域相互作用来增强 RNase E 活性，这代表了 RNase E 激活的新机制。相比之下，RNA 结合具有招募作用，增加了形成生产性 RapZ/GlmZ/RNase E 复合物的可能性。