Minagawa Asako, Takaku Hiroaki, Takagi Masamichi, Nashimoto Masayuki
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niitsu, Niigata 956-8603, Japan.
J Biol Chem. 2004 Apr 9;279(15):15688-97. doi: 10.1074/jbc.M313951200. Epub 2004 Jan 27.
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.
tRNA的3'-末端CCA序列对于tRNA的氨酰化作用以及在核糖体上的翻译过程至关重要。tRNA最初转录为包含5'和3'额外序列的较大前体分子。在没有编码CCA的tRNA中，鉴别核苷酸后的3'额外序列通常会被tRNA 3'加工内切核糖核酸酶（3' tRNase，或核糖核酸酶Z）切除，然后在其上添加3'-末端CCA残基。在这里，我们使用来自海栖热袍菌（Thermotoga maritima）的各种前体tRNA分析了海栖热袍菌3' tRNase的酶学特性，其中46个tRNA基因中只有一个例外，其余均编码CCA。我们发现该酶具有前所未有的活性，能够精确地在CCA序列之后而非鉴别核苷酸之后切割含CCA的前体tRNA。对前体tRNA变体的分析表明，核苷酸75和76处的CA残基是该酶在核苷酸76处的A之后切割前体tRNA所必需的，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，作为海栖热袍菌中唯一没有编码CCA的前体tRNA，前体tRNA(Met)是在鉴别核苷酸之后被切割的。动力学数据表明海栖热袍菌3' tRNase中存在一个CCA结合结构域。我们还鉴定了两个对切割位点选择至关重要的氨基酸残基以及几个对催化作用必不可少的残基。对来自另一种真细菌大肠杆菌以及两种古细菌嗜热栖热菌（Thermoplasma acidophilum）和气生嗜热栖热菌（Pyrobaculum aerophilum）的3' tRNase切割位点的分析证实了这两个氨基酸残基对切割位点选择的重要性。
