Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
Cells. 2022 May 2;11(9):1522. doi: 10.3390/cells11091522.
The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions.
细菌酶 tRNA 2-硒代尿苷合酶（SelU）负责将某些细菌 tRNA 反密码子中的 5-取代 2-硫代尿苷（R5S2U）转化为 5-取代 2-硒代尿苷（R5Se2U）。我们已经使用合成 RNA 证明，S2U→Se2U 的转化是一个两步过程，其中 S2U-RNA 香叶化，得到的 geS2U-RNA 被硒化。目前的问题是 SelU 如何识别其底物，以及天然 tRNA 中 R5S2U→R5Se2U 转化的细胞途径是什么。在本研究中，我们表征了 SelU 的底物要求，鉴定了 SelU 相关的 tRNA 及其在摆动位置的特异性修饰。最后，我们解释了 tRNA 硒化的步骤顺序。RNA 链中的 S2U 位置、修饰的侧翼序列和 RNA 底物的长度都对 SelU 的识别有关键影响。SelU 对个别 RNA 亲和力的 MST 数据证实了这一推测过程。SelU 结合 R5S2U-tRNA，然后催化其香叶化为 R5geS2U-tRNA，该 RNA 仍与酶结合，并在转化的下一步被硒化。最后，R5Se2U-tRNA 离开酶并参与翻译过程。酶不会直接催化 R5S2U-tRNA 的硒化，而 R5geS2U-tRNA 是反应线性序列中的中间产物。
