Oommen A, Li X Q, Gegenheimer P
Department of Botany, University of Kansas, Lawrence 66045-2106.
Mol Cell Biol. 1992 Feb;12(2):865-75. doi: 10.1128/mcb.12.2.865-875.1992.
tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.
真核细胞核和细胞器中的tRNA以前体形式合成，这些前体缺乏3'-末端CCA序列，并具有5'（前导序列）和3'（尾随序列）延伸。因此，3'尾随序列的核酸酶切割和CCA的添加是功能性tRNA 3'末端形成所必需的。许多叶绿体tRNA基因在第74位编码一个C，该C在加工过程中不会被去除，但可以作为CCAOH末端的第一个碱基掺入。然而，核苷酸74下游的序列总是被去除。用粗制或部分纯化的叶绿体酶组分处理含有完整CCA74 - 76序列的合成酵母前体tRNA(Phe)底物。3'-延伸的底物（tRNA-CCA-尾随序列）仅在核苷酸74和75之间被切割，产生tRNA-COH，而3'-成熟转录本（tRNA-CCAOH）根本不被切割。一个5'、3'-延伸的叶绿体tRNA-CAG-尾随序列也完全被加工成tRNA-COH。此外，一个5'-成熟、3'-延伸的酵母前体tRNA(Phe)衍生物，tRNA-ACA-尾随序列，其中C74被A取代，在A74之后被精确切割。相比之下，我们发现从小麦胚中部分纯化的酶组分（一种核/细胞质活性）在核苷酸73和74之间切割3'-延伸的酵母tRNA(Phe)前体，产生tRNA(OH)。这种特异性与所有先前表征的核酶制剂的特异性一致。我们得出结论：(i)叶绿体tRNA 3'-加工内切核酸酶在核苷酸74之后切割，而不考虑周围序列的性质；(ii)这种特异性不同于植物核/细胞质加工核酸酶的特异性，后者在碱基73之后切割；(iii)由于3'-成熟tRNA不是这两种活性的底物，这些3'核酸酶必须需要具有延伸超过核苷酸76的3'-末端延伸的底物。这种底物特异性可能会防止成熟tRNA被3'加工系统进行适得其反的切割。
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