Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA
J Bacteriol. 2019 Sep 20;201(20). doi: 10.1128/JB.00433-19. Print 2019 Oct 15.
In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (moU) or 5-methoxycarbonylmethoxyuridine (mcmoU). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (hoU) remain unknown. Here, a number of genes in and are identified that are required for wild-type (wt) levels of hoU. The operon is identified in as important for the biosynthesis of hoU. Both and are homologs to peptidase U32 family genes, which includes the gene required for hoC synthesis in Deletion of either or , or both, gives a 50% reduction in moU tRNA levels. In , was found to be the only one of four peptidase U32 genes involved in hoU synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmoU as that observed for moU in the mutants. By analyzing the genomic context of homologs, the ferredoxin YfhL is shown to be required for hoU synthesis in to the same extent as Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that hoU biosynthesis in bacteria is similar to that of hoC, but additional genes and substrates are required for complete modification. Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both and These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.
在细菌中，反密码子第 34 位为尿嘧啶的 tRNA 可解码四联体简并家族密码子，通常会被 5-甲氧基尿嘧啶（moU）或 5-甲氧基羰基甲氧基尿嘧啶（mcmoU）修饰。这些修饰对于在摆动位置扩展识别一些密码子至关重要。虽然这些修饰的烷基化步骤已经被描述过，但是编码 U34 羟化为 5-羟尿嘧啶（hoU）的基因仍然未知。在这里，鉴定出了 和 中许多与野生型（wt）hoU 水平相关的基因。鉴定出 操纵子在 中对 hoU 的生物合成很重要。 和 都是肽酶 U32 家族基因的同源物，其中包括 基因，该基因在 中负责 hoC 的合成。缺失 或 或两者都会导致 moU tRNA 水平降低 50%。在 中，发现只有四个肽酶 U32 基因中的 一个参与 hoU 的合成。有趣的是，该突变体显示与 moU 突变体相同的 50%的(m)cmoU 降低。通过分析 同源物的基因组背景，发现铁氧还蛋白 YfhL 与 一样，是 hoU 在 中合成所必需的。铁硫生物合成和预苯酸生物合成所需的其他基因也会使修饰降低 50%。总的来说，这些数据表明细菌中 hoU 的生物合成与 hoC 相似，但需要额外的基因和底物才能完成修饰。tRNA 中的修饰核苷酸有助于优化其结构和功能，以实现遗传密码的准确翻译。这些修饰的生物合成是揭示细胞中独特生物化学和代谢的肥沃土壤。在这项工作中，在 和 中鉴定了一些 tRNA 中摆动位置的尿嘧啶的新型厌氧羟化所需的基因。这些基因编码铁硫簇蛋白，它们的缺失使两种生物体内的羟尿嘧啶水平降低 50%。铁硫簇和预苯酸生物合成所需的其他基因和以前描述的铁氧还蛋白基因都显示出类似的羟尿嘧啶水平降低，这表明仍需要其他基因进行修饰。
