MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli
MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli
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DOI：
10.1021/bi026536
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发表时间：
2003-02-04
期刊：
影响因子：
2.9
通讯作者：
Lauhon, CT
中科院分区：
文献类型：
--
作者：
Kambampati, R;Lauhon, CT
硫代糖苷是tRNA中唯一存在的。在许多生物体中，对赖氨酸、谷氨酸和谷氨酰胺具有特异性的tRNA在摆动位置34处含有高度修饰的2-硫代尿苷（s（2）U）衍生物。s（2）U34的s（2）基团稳定反密码子结构，赋予核糖体与tRNA结合的能力并改善阅读框维持。早期的研究已经定位了mnmA基因（以前称为asuE或trmU），后来确定其为大肠杆菌中s（2）U修饰所必需。我们已经制备了mnmA基因的非极性缺失，并表明它不是E.杆菌我们还从E.大肠杆菌中，并过量生产和纯化的蛋白。使用凝胶迁移率变动分析，我们表明MnmA结合未修饰的E。colitRNA（Lys）具有低微摩尔范围内的亲和力。MnmA不与非底物E显著结合。coli tRNA（Phe）。证实了这一点，tRNA（Glu）保护MnmA免受胰蛋白酶消化。ATP还保护MnmA免受胰蛋白酶水解，这表明存在与氨基酸序列分析一致的ATP结合位点。我们用未修饰的E. coli tRNA（Glu）为底物。该活性需要MnmA、Mg-ATP、L-半胱氨酸和半胱氨酸脱硫酶IscS。使用[S-35]半胱氨酸对巯基化tRNA酶进行HPLC分析，证实体外反应产物为s（2）U。与4-硫尿苷合成的情况一样，纯化的Iscs-过硫化物能够在不存在半胱氨酸的情况下为体外s（2）U合成提供硫。代表tRNAGlu和tRNALys反密码子茎环的小RNA是与全长tRNA活性相当的底物，这表明底物识别的主要决定因素包含在该区域内。
Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group Of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis Of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, L-Cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [S-35]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNAGlu and tRNALys are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.