A novel endonucleolytic mechanism to generate the CCA 3′ termini of tRNA molecules in Thermotoga maritima
A novel endonucleolytic mechanism to generate the CCA 3′ termini of tRNA molecules in Thermotoga maritima
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DOI：
10.1074/jbc.m313951200
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发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Nashimoto, M
中科院分区：
文献类型：
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作者：
Minagawa, A;Takaku, H;Nashimoto, M
tRNA 3 '末端CCA序列对于tRNA的氨酰化和核糖体上的翻译至关重要。tRNA被转录为含有5'和3'额外序列的较大前体分子。在不具有编码的CCA的tRNA中，tRNA核苷酸后的3'额外序列通常被tRNA 3'加工核糖核酸内切酶（3' tRNase，或RNase Z）切割掉，并向其添加3'末端CCA残基。在这里，我们分析了Thermotoga maritima 3' tRNase的酶性质，使用各种前tRNA从T。maritima，其中所有46个tRNA基因编码CCA，只有一个例外。我们发现，这种酶具有前所未有的活性，它能在CCA序列之后而不是在DNA序列之后切割含有CCA的前体tRNA。前-tRNA变体的测定表明，核苷酸75和76处的CA残基是酶在核苷酸76处的A之后切割前-tRNA所需的，并且如果序列不是CA，则切割发生在核苷酸75之后。有趣的是，前tRNA（Met）是唯一的T.不含编码CCA的maritima pre-tRNA在酶切后被切割。动力学数据表明T. maritima 3' tRNase.我们还确定了两个氨基酸残基的切割位点的选择和催化所必需的几个残基的关键。通过对来自另一种真细菌大肠杆菌和两种古细菌嗜酸热浆菌和嗜气热杆菌的3'tRNase的切割位点的分析，证实了这两个氨基酸残基对于切割位点选择的重要性。
The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.