The 3′ end CCA of mature tRNA is an antideterminant for eukaryotic 3′-tRNase
The 3′ end CCA of mature tRNA is an antideterminant for eukaryotic 3′-tRNase
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DOI：
10.1017/s1355838299981256
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发表时间：
1999-02-01
期刊：
影响因子：
4.5
通讯作者：
Levinger, L
中科院分区：
文献类型：
--
作者：
Mohan, A;Whyte, S;Levinger, L
细胞质tRNA在真核细胞核中经历转录后5'和3'末端加工，并且CCA（其形成所有tRNA的成熟3'末端）必须在tRNA可以被氨酰化并用于翻译之前通过tRNA核苷酸转移酶添加。真核3 '-tRNase可以通过在3'端碱基（受体茎的最后一个碱基对的3'端的未配对核苷酸）的3'侧上切割来内切核酸去除3'端尾部。尽管3'末端尾部的长度和序列范围很广，但该反应仍在进行，只是含有3'末端CCA的成熟tRNA不是小鼠3 '-tRNase的底物（Nashimoto，1997，Nucleic Acids Res 25：1148-1154）。在此，我们以果蝇tRNA（His）为底物，用果蝇和猪3 '-tRNase扩展了这一结果。我们还测试了一系列终止于tRNA碱基（-）的tRNA，其中添加了一个C（+C），添加了两个Cs（+CC），以及添加了CCA（+CCA）作为3 '-tRNase抑制剂。抑制作用与果蝇和猪3 '-tRNase都是竞争性的。3 '-tRNase反应的产物（-）是良好的3'-tRNase抑制剂，其K-1约为正常3 '-tRNase底物的K-M的两倍。K-I随着超过tRNA碱基添加的每个核苷酸而增加，直到当tRNA+CCA用作抑制剂时，K-I大约是底物K-M的40倍。因此，3 '-tRNase可以保持自由以加工具有3'末端尾部的前体，因为它几乎不被tRNA+CCA抑制，从而确保tRNA可以进行氨酰化。3 '-tRNase的活性位点可能已经进化为与tRNA+CCA特别不匹配。
Cytoplasmic tRNAs undergo posttranscriptional 5' and 3' end processing in the eukaryotic nucleus, and CCA (which forms the mature 3' end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacylated and utilized in translation. Eukaryotic 3'-tRNase can endonucleolytically remove a 3' end trailer by cleaving on the 3' side of the discriminator base (the unpaired nucleotide 3' of the last base pair of the acceptor stem). This reaction proceeds despite a wide range in length and sequence of the 3' end trailer, except that mature tRNA containing the 3' terminal CCA is not a substrate for mouse 3'-tRNase (Nashimoto, 1997, Nucleic Acids Res 25:1148-1154). Herein, we extend this result with Drosophila and pig 3'-tRNase, using Drosophila melanogaster tRNA(His) as substrate. Mature tRNA is thus prevented from recycling through 3' end processing.We also tested a series of tRNAs ending at the discriminator base (-), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3'-tRNase inhibitors. Inhibition was competitive with both Drosophila and pig 3'-tRNase. The product of the 3'-tRNase reaction (-) is a good 3'-tRNase inhibitor, with a K-l approximately two times K-M for the normal 3'-tRNase substrate. K-l increases with each nucleotide added beyond the discriminator base, until when tRNA+CCA is used as inhibitor, K-l is approximately forty times the substrate K-M. The 3'-tRNase can thus remain free to process precursors with 3' end trailers because it is barely inhibited by tRNA+CCA, ensuring that tRNA can progress to aminoacylation. The active site of 3'-tRNase may have evolved to make an especially poor fit with tRNA+CCA.