RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

中文翻译：

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阐明詹氏甲烷球菌 RNase P（一种多亚基催化核糖核蛋白）的结构与功能关系

RNase P 是一种核糖核蛋白 (RNP)，可催化生命所有领域中前体 tRNA 中 5' 前导序列的去除。最近一项对詹氏甲烷球菌 (Mja) RNase P 的冷冻电镜研究产生了一个二聚体结构的 4.6-Å 分辨率模型，其中每个全酶单体包含一个 RNase P RNA (RPR) 和 5 个 RNase P 蛋白 (RPP) 各一个拷贝；POP5、RPP30、RPP21、RPP29、L7Ae）。在这里，我们使用天然质谱 (MS)、质量光度测定 (MP) 和生化实验来 (i) 在体外验证 Mja RNase P 全酶的寡聚状态，(ii) 为每个全酶单体找到不同的化学计量两个 L7Ae 拷贝，以及 (iii) 评估两个 L7Ae 拷贝对于最佳切割活性是否是必需的。通过突变 RPR 中的所有扭结转角，我们发现废除规范的 L7Ae-RPR 相互作用对于 RNase P 组装和功能并不有害，因为 L7Ae 和其他 RPP 之间的蛋白质-蛋白质相互作用提供了冗余。我们的结果为 RNase P 的结构和进化提供了新的见解，并强调了天然 MS 和 MP 在综合结构生物学方法中的效用，这些方法旨在增强从低/中分辨率冷冻电镜模型中获得的信息。