Laboratory of Biophysical Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Nishi-ku, Fukuoka, Japan.
Laboratory of Biophysical Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Nishi-ku, Fukuoka, Japan.
J Biol Chem. 2021 Sep;297(3):101028. doi: 10.1016/j.jbc.2021.101028. Epub 2021 Jul 31.
Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.
核糖核酸酶 P（RNase P）是一种内切核酸酶，可催化前体 tRNA（pre-tRNA）5' 前导序列的加工。真核生物中的核糖核蛋白 RNase P 和仅含蛋白质的 RNase P（PRORP）已得到广泛研究，但原核核酸酶识别和切割 pre-tRNA 的机制尚不清楚。为了深入了解这一机制，我们研究了 Aquifex RNase P（HARPs）的同源物，这些酶被认为是大约 23 kDa 的酶，仅由该核酸酶结构域组成。我们确定了第一个鉴定出的 HARP 酶 Aq880 的冷冻电镜结构。出乎意料的是，该结构揭示 Aq880 由核酸酶和突出螺旋（PrH）结构域组成。Aq880 单体通过 PrH 结构域组装成二聚体。六个二聚体形成具有左手一转超螺旋结构的十二聚体。该结构还揭示了 Aq880 的活性位点类似于真核 PRORPs。pre-tRNA 对接模型表明，pre-tRNA 的 5' 加工是通过十二聚体中的两个相邻二聚体实现的。一个二聚体负责催化，另一个二聚体的 PrH 结构域负责识别 pre-tRNA 弯曲处。我们的研究表明，HARPs 从 pre-tRNA 弯曲处测量到恒定距离以切割 5' 前导序列，这类似于真核 PRORPs 和核糖核蛋白 RNase P 的机制。总的来说，这些发现阐明了不同类型的 RNase P 酶如何利用相同的 pre-tRNA 加工。
