Catalysis by an "RNA-free" RNase P

“无 RNA”RNase P 的催化作用

• 批准号: 131747664
• 负责人: Professor Dr. Roland K. Hartmann
• 金额: --
• 依托单位: Center of Anatomy & Cell Biology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/131747664?language=en
• 关键词: Catalysis; RNA; free; RNase

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5 ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized until just recently are composed of a catalytic RNA plus one or more proteins, and tRNA 5 end maturation was thus generally considered a universal ribozymecatalyzed process. We recently overcame this paradigm when we identified the components of human mitochondrial RNase P (mtRNase P), finding only proteins, and reconstituted the enzymatic activity using just three of these. Evidently, a complex of three proteins replaced the ribozyme-remnant from the hypothetical RNA world during the evolution of animal mitochondria. This protein enzyme nonetheless drives the same biochemical reaction and fulfills the same biological function as its RNA-based predecessor. The RNase P family appears thus unique in biology as it includes isoforms representing partial as well as complete evolutionary transitions from RNA- to protein-based catalysis. In this project we aim to elucidate the catalytic mechanism of mtRNase P, and to compare the catalytic strategy of this protein enzyme to that of its more common ribozymal isoforms. mtRNase P is composed of three proteins, termed MRPP1-3, at least two of which are involved in other tRNA-related and -unrelated biochemical pathways too. MRPP1 and MRPP2 constitute a methyltransferase, involved in the modification of mitochondrial tRNAs. They also appear to confer tRNA-specificity to mtRNase P, but only upon addition of the third protein, MRPP3, and magnesium, tRNA 5’ end cleavage can be reconstituted. MRPP3 contains putative RNA-binding and metallonuclease domains and it is thus reasonable to assume that it is the actual nuclease moiety of the enzyme. By determining (1) the atomic scale structure of MRPP3, its molecular interactions with the other components of mtRNase P and with tRNA precursors, by dissecting (2) functional groups of enzyme and substrate involved in substrate recognition, cleavage site positioning, and catalysis, by studying (3) the role and interactions of metal ions involved in hydrolysis, and by studying (4) the cell biology and evolution of MRPP3, we aim to obtain a comprehensive understanding of the catalytic mechanism and biology of mtRNase P. Ultimately, we may learn how and why evolution replaced an ancient RNA catalyst in the animal mitochondrial lineage, while it retained ribozyme catalysis in the vast majority of phylogenetic domains.

tRNA作为未成熟的前体合成，并且在其功能成熟的过程中，在其5端的额外核苷酸被称为RNase P的内切核酸酶去除。直到最近才表征的所有RNase P酶都由催化RNA加上一种或多种蛋白质组成，并且tRNA 5端成熟因此通常被认为是通用的核酶催化过程。我们最近克服了这种范式，当我们确定了人类线粒体RNase P（mtRNase P）的组成部分时，只发现了蛋白质，并仅使用其中的三种重建了酶活性。显然，在动物线粒体的进化过程中，一个由三种蛋白质组成的复合体取代了来自假设的RNA世界的核酶残余物。尽管如此，这种蛋白酶仍然驱动相同的生化反应，并实现与其基于RNA的前身相同的生物学功能。因此，RNase P家族在生物学上是独特的，因为它包括代表从RNA到蛋白质催化的部分和完全进化转变的同种型。在这个项目中，我们的目标是阐明mtRNase P的催化机制，并比较这种蛋白酶的催化策略，其更常见的核酶异构体。mtRNase P由三种蛋白质组成，称为MRPP 1 -3，其中至少两种也参与其他tRNA相关和不相关的生化途径。MRPP 1和MRPP 2构成甲基转移酶，参与线粒体tRNA的修饰。它们似乎也赋予mtRNase P tRNA特异性，但只有在加入第三种蛋白质MRPP 3和镁后，才能重建tRNA 5'端切割。MRPP 3含有推定的RNA结合和金属核酸酶结构域，因此可以合理地假设它是该酶的实际核酸酶部分。通过确定（1）MRPP 3的原子尺度结构，其与mtRNase P的其他组分和与tRNA前体的分子相互作用，通过解剖（2）参与底物识别、切割位点定位和催化的酶和底物的官能团，通过研究（3）参与水解的金属离子的作用和相互作用，通过研究（4）MRPP 3的细胞生物学和进化，我们旨在全面了解mtRNase P的催化机制和生物学。最终，我们可以了解进化如何以及为什么在动物线粒体谱系中取代了古老的RNA催化剂，而它在绝大多数系统发育域中保留了核酶催化作用。