Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System*
Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System*
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DOI：
10.1074/jbc.272.1.255
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发表时间：
1997-01
期刊：
影响因子：
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通讯作者：
Zhong Wang;M. Sachs
中科院分区：
文献类型：
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作者：
Zhong Wang;M. Sachs
在同源、无细胞的体外翻译系统中，重组了粗糙脉孢菌arg-2 mRNA 5′前导序列上游开放阅读框（uORF）介导的翻译控制。无细胞N. crassa系统的开发，需要帽和多聚（A）的存在下，RNA的最大翻译，这是氨基酸依赖性。24个密码子的arg-2 uORF位于加帽和腺苷酸化的合成荧光素酶RNA的5′-前导区时，在该系统中赋予Arg特异性负调控。改善uORF翻译起始上下文降低了荧光素酶的产生，并且仅略微增加了Arg特异性调节的幅度。uORF Asp密码子12突变为Asn，消除了体内Arg特异性调节，消除了体外调节。uORF翻译起始密码子的消除也消除了Arg特异性调节。精氨酸特异性调节在体外似乎是可逆的。RNA稳定性的控制似乎不是体外Arg特异性调节的主要组成部分。将Arg加入体外翻译反应中与加入Arg相关化合物的效果比较表明，Arg特异性翻译调节对L-精氨酸具有特异性。
Translational control mediated by an upstream open reading frame (uORF) in the 5′-leader of the Neurospora crassa arg-2 mRNA was reconstituted in a homologous, cell-free in vitro translation system. A cell-free N. crassa system was developed that required the presence of cap and poly(A) on RNA for maximal translation and that was amino acid-dependent. The 24-codon arg-2 uORF, when placed in the 5′-leader region of capped and adenylated synthetic luciferase RNAs, conferred Arg-specific negative regulation in this system. Improving the uORF translation initiation context decreased luciferase production and only slightly increased the magnitude of Arg-specific regulation. Mutation of uORF Asp codon 12 to Asn, which eliminates Arg-specific regulation in vivo, eliminated regulation in vitro. Elimination of the uORF translation initiation codon also eliminated Arg-specific regulation. Arg-specific regulation in vitro appeared to be reversible. Control of RNA stability did not appear to be a primary component of Arg-specific regulation in vitro. Comparison of the effects of adding Arg to in vitro translation reactions with adding compounds related to Arg indicated that Arg-specific translational regulation was specific for L-arginine.