Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis
Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis
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DOI：
10.1128/jb.02625-14
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发表时间：
2015-06-01
影响因子：
3.2
通讯作者：
Dos Santos, Patricia C.
中科院分区：
文献类型：
--
作者：
Black, Katherine A.;Dos Santos, Patricia C.
谷氨酸、谷氨酰胺和赖氨酸tRNA分子中摆动位置的2-硫代尿苷（s（2）U）修饰用于稳定反密码子结构，改善核糖体结合和翻译过程的总体效率。大肠杆菌中s（2）U的生物合成需要一个半胱氨酸脱硫酶（IscS）、一个硫脲苷酶（MnmA）和五个中间硫传递酶（TusABCDE）。急诊coli MnmA使tRNA腺苷酸化并随后硫醇化以形成s（2）U修饰。枯草芽孢杆菌缺乏IscS和中间硫中继蛋白，但其基因组含有直接邻近mnmA的半胱氨酸脱硫酶基因yrvO。yrvO和mnmA的基因组同线性结合Tus蛋白的缺失表明这些蛋白在s（2）U形成中的潜在功能。在这里，我们提供的证据表明，B。subtilis YrvO和MnmA是s（2）U生物合成的充分条件。条件B。枯草杆菌敲除菌株中的s（2）U丰度与MnmA表达相关，在E. coli IscS或MnmA缺陷菌株的研究表明，这些蛋白在s（2）U生物合成中具有竞争力。体外实验证明了由YrvO和MnmA形成s（2）U，动力学分析建立了B之间的伙伴关系。枯草杆菌蛋白质，取决于ATP的存在。此外，我们观察到E.用B回收与s（2）U耗尽相关的大肠杆菌Delta iscS和Delta mnmA菌株。subtilis yrvO和mnmA。这些结果支持的建议，参与一个专门的半胱氨酸脱硫酶，YrvO，在s（2）U合成绕过需要一个复杂的生物合成途径，通过直接硫转移到MnmA.IMPORTANCEThe 2-thiouridine（s（2）U）修饰的摆动位置在谷氨酸，谷氨酰胺和赖氨酸tRNA是保守的，在所有三个领域的生活和稳定的反密码子结构，从而保证翻译的保真度。大肠杆菌中s（2）U的生物合成需要七种蛋白质：半胱氨酸脱硫酶IscS、硫脲苷酶MnmA和五种中间硫传递酶（TusABCDE）。枯草芽孢杆菌和大多数革兰氏阳性菌缺乏一套完整的生物合成组分。有趣的是，mnmA编码序列位于邻近yrvO，编码半胱氨酸脱硫酶。在这项工作中，我们提供的证据表明，B。subtilis YrvO能够将硫直接转移至MnmA。这两种蛋白质都足以使s（2）U在一条独立于E.杆菌
The 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS-or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli Delta iscS and Delta mnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s(2)U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.