Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP
Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP
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DOI：
10.1101/gad.12.11.1678
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发表时间：
1998-06-01
影响因子：
10.5
通讯作者：
Engelke, DR
中科院分区：
文献类型：
--
作者：
Chamberlain, JR;Lee, Y;Engelke, DR
核糖核酸酶P （RNase P）是一种核糖核蛋白酶，可切割前体tRNA转录物以获得成熟的5'端。真细菌中的RNase P具有一个大的催化RNA亚基和一个小的蛋白质亚基，这是体内前体tRNA切割所必需的。尽管真核全酶具有相似的大RNA亚基，但先前在许多系统中的研究表明，真核全酶需要更高的蛋白质含量。我们纯化了酿酒酵母核核糖核酸酶P，使其具有明显的同质性，从而首次对出乎意料的复杂亚基组成进行了全面分析。通过离子阱质谱法进行多肽测序，鉴定出9种与核RNase P RNA亚基共聚合的蛋白质，比细菌酶中的蛋白质总数多20倍。所有这些蛋白质都是由RNase P活性和细胞活力所必需的基因编码的。先前的遗传研究表明，四种蛋白质可能是RNase P和RNase MRP的亚基，RNase MRP是相关的rRNA加工酶。我们证明，所有这四种蛋白质，Pop1p, Pop3p， Pop4p和Rpp1p，都是RNase P的完整亚基。此外，五个新鉴定的蛋白质亚基中的四个，Pop5p, Pop6p， Pop7p和Pop8p，似乎也在RNase P和RNase MRP之间共享。通过基因耗尽和免疫沉淀研究，RNase P全酶只有一种多肽Rpr2p是独特的。真核生物RNase P蛋白亚基数量的大量增加与真核生物功能复杂性的增加是一致的。核RNase P和RNase MRP的结构相似性表明，它们在前trna和前rrna加工途径中的某些功能可能重叠或协调。
Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5' ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme. All of these proteins are encoded by genes essential for RNase P activity and for cell viability. Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme. We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P. In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP. Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies. The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes. The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.