Dead box Rh1B RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E
Dead box Rh1B RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E
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DOI：
10.1074/jbc.m206618200
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发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Sue, LC
中科院分区：
文献类型：
--
作者：
Liou, GG;Chang, HY;Sue, LC
大肠杆菌 RNA 降解体是一种多组分核糖核酸水解复合物，由三种主要蛋白质组成，这些蛋白质组装在由核酸内切酶 RNase E 的 C 端区域提供的支架上。使用大肠杆菌双杂交系统和 BIAcore 装置，我们研究了三种蛋白质：多核苷酸磷酸化酶 (PNPase)、Rh1B RNA 解旋酶和烯醇化酶（一种糖酵解蛋白）的能力。独立于 RNase E 进行物理和功能上的相互作用。在此，我们报道了 Rh1B 在体外和体内都可以与 PNPase 物理结合，并且还可以与其自身形成同型二聚体。然而，在相同条件下未检测到 Rh1B 或 PNPase 与烯醇酶的结合。 BIAcore 分析揭示了 Rh1B 单元之间双分子相互作用以及 Rh1B 与 PNPase 相互作用的实时、直接结合。此外，在没有 RNase E 的情况下，纯化的 Rh1B 可以进行双链 RNA 的 ATP 依赖性解旋，从而与 PNPase 的核酸外切酶活性一起调节双链 RNA 的降解。这些结果首次提供了证据，表明在没有 RNase E 的情况下，各个降解体蛋白组分的功能和物理相互作用都可以发生，并提出了这样的前景：在体内检测到的不依赖于 RNase E 的 Rh1B RNA 解旋酶和 PNPase 复合物可能构成微型机器，在物理上与多组分 RNA 降解体不同的结构中协助双链体 RNA 的降解。
The Escherichia coli RNA degradosome is a multicomportent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), Rh1B RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here ;we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of Rh1B or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the Rh1B interaction with PNPase. Furthermore, in the absence of RNase E, purified Rh1B can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of Rh1B RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.