Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, Durham, NC, USA.
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, Durham, NC, USA.
Nat Struct Mol Biol. 2019 Sep;26(9):830-839. doi: 10.1038/s41594-019-0289-8. Epub 2019 Sep 5.
Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.
核糖体组装是一个复杂的过程，依赖于转译酶的协调作用，以产生功能性核糖体亚基并确保细胞的翻译能力。内切核酸酶（RNase）Las1 和多核苷酸激酶（PNK）Grc3 组装成多酶复合物，在此称为 RNase PNK，以协调前体核糖体 RNA（rRNA）的加工。RNase PNK 属于功能多样的 HEPN 核酸酶超家族，其成员依赖于不同的线索来激活核酸酶。为了确定 RNase PNK 如何协调其双重酶活性，我们解决了一系列嗜热毛壳菌 RNase PNK 在多种构象状态下的冷冻电镜结构。这些结构揭示了 RNase PNK 采用蝴蝶样的结构，包含一个复合的 HEPN 核酸酶活性位点，两侧是离散的 RNA 激酶位点。我们确定了两个分子开关，它们协调核酸酶和激酶的功能。总之，我们的结构和相应的功能研究建立了一种新的 HEPN 核酸酶激活机制，这对于核糖体的产生是必不可少的。
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