Minimum requirements for substrates of mammalian tRNA 3′ processing endoribonuclease
Minimum requirements for substrates of mammalian tRNA 3′ processing endoribonuclease
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DOI：
10.1021/bi9911942
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发表时间：
1999-09-14
期刊：
影响因子：
2.9
通讯作者：
Kaspar, RL
中科院分区：
文献类型：
--
作者：
Nashimoto, M;Tamura, M;Kaspar, RL
哺乳动物tRNA 3'加工核糖核酸内切酶（3' tRNase）从前体tRNA中除去3'端核苷酸后的一个3'尾部为了阐明对3' tRNase底物的最低要求，我们测试了缺乏D和反密码子茎环结构域的小前tRNA（Arg）底物用于被纯化的猪3' tRNase切割。小前tRNA（R-ATW）由受体茎，额外环，T茎-环结构域、3 ′核苷酸和3 ′尾部比全长野生型更有效地被切割。三个R-ATW衍生物的催化效率也高于全长野生型的催化效率，所述R-ATW衍生物被构建为破坏原始T茎碱基对。猪3' tRNase有效地加工由T茎环结构域、受体茎、3'核苷酸和3'尾部组成的“微螺旋”（R-ATM 5），而该酶从不切割由T环、受体茎、3'核苷酸和3'尾部组成的“微螺旋”。在T环中具有1至7个碱基取代的5种R-ATM 5衍生物都比全长野生型稍微更有效地切割，并且比R-ATM 5稍微更不有效地切割。比微型螺旋小一个碱基对的A螺旋（“微型螺旋Delta 1”）是良好的底物，而含有连续的10个碱基对茎的小螺旋是差的底物。这三种小底物的切割发生在酶切位点之后，并在酶切位点下游的一到三个核苷酸处。从这些结果，我们得出结论，哺乳动物3' tRNase有效切割的最低底物是微螺旋或微螺旋Delta 1，其中似乎没有必需的碱基。
Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA), To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase, A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5, A helix ("minihelix Delta 1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelices Delta 1, in which there seem to be no essential bases.