Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada; Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada.
J Biol Chem. 2023 May;299(5):104612. doi: 10.1016/j.jbc.2023.104612. Epub 2023 Mar 16.
Among the large and diverse collection of tRNA modifications, 7-methylguanosine (mG) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNA enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of WT and single substitution variants of TrmB with tRNA. Our results reveal the role of S-adenosylmethionine for rapid and stable tRNA binding, the rate-limiting nature of mG46 catalysis for tRNA release, and the importance of residues R26, T127, and R155 across the entire surface of TrmB for tRNA binding.
在大量多样的 tRNA 修饰中，7-甲基鸟苷（mG）经常在 tRNA 可变环的第 46 位发现。这种修饰由 TrmB 酶引入，该酶在细菌和真核生物中保守。然而，TrmB 识别 tRNA 的分子决定因素和机制尚不清楚。补充了不同缺乏 TrmB 同源物的生物体的各种表型报告，我们在这里报告大肠杆菌 ΔtrmB 敲除菌株对过氧化氢的敏感性。为了实时深入了解大肠杆菌 TrmB 与 tRNA 结合的分子机制，我们开发了一种新的测定法，该测定法基于在体外转录的 tRNA 的第 8 位引入 4-硫尿嘧啶修饰，使我们能够对未修饰的 tRNA 进行荧光标记。使用该荧光 tRNA 的快速动力学停止流动测量，我们检查了 WT 和 TrmB 的单取代变体与 tRNA 的相互作用。我们的结果揭示了 S-腺苷甲硫氨酸对快速和稳定的 tRNA 结合的作用，mG46 催化对 tRNA 释放的限速性质，以及 TrmB 整个表面上的残基 R26、T127 和 R155 对 tRNA 结合的重要性。
