Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF
Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF
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DOI：
10.1074/jbc.m116.747865
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发表时间：
2016-10-14
影响因子：
4.8
通讯作者：
Limbach, Patrick A.
中科院分区：
文献类型：
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作者：
Addepalli, Balasubrahmanyam;Limbach, Patrick A.
假尿苷存在于几乎所有的细胞核糖核酸（RNA）中。在假尿苷的多种特征中，使信使RNA（mRNA）高度可翻译和非免疫原性是直接涉及蛋白质合成中的这种修饰的一个这样的特征。我们报告存在的假尿苷在反密码子的大肠杆菌酪氨酸转移RNA（tRNA）的位置35。假尿苷通过多种检测方法进行验证，包括假尿苷特异性化学衍生化和液相色谱串联质谱（LC-MS/MS）过程中RNA的气相解离。从E.大肠杆菌假尿苷合酶敲除突变体鉴定RluF为负责该修饰的酶。此外，当荧光素酶报告基因编码序列之前有多个酪氨酸密码子时，这种修饰的缺乏损害了荧光素酶报告基因编码序列的翻译能力。这种效应对细菌表达系统中富含酪氨酸密码子的mRNA的翻译具有影响。
Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.