University of Lethbridge, Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, Lethbridge, AB T1K 3M4, Canada.
Nucleic Acids Res. 2020 Aug 20;48(14):7981-7990. doi: 10.1093/nar/gkaa548.
tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA's D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA's tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.
tRNAs 是所有细胞中修饰程度最高的 RNA，T 臂 54 位的 5-甲基尿嘧啶（m5U）的形成是所有 tRNA 中常见的 RNA 修饰。这种 m5U 修饰是由甲基转移酶 TrmA 生成的。在这里，我们通过实验验证了大肠杆菌 TrmA 具有双重功能的假设，它既可以作为甲基转移酶，也可以作为 tRNA 伴侣。我们鉴定了 TrmA 的 RNA 结合域中的两个保守残基 F106 和 H125，它们与 tRNA 弯角相互作用，对于 tRNA 结合至关重要。共培养竞争测定显示，TrmA 的催化活性对于细胞活力很重要，并且 F106 或 H125 的取代会损害细胞活力。我们直接表明，TrmA 可以独立于其催化活性增强体外 tRNA 的折叠。总之，我们的研究表明，TrmA 的 RNA 结合域中的 F106 和 H125 充当楔子，破坏 tRNA 的 D 臂和 T 臂之间的三级相互作用；这种 tRNA 展开是 TrmA 的 tRNA 伴侣活性的机制基础。除了假尿嘧啶合酶 TruB 之外，TrmA 是第二种被证明具有 tRNA 伴侣活性的 tRNA 修饰酶，它支持 RNA 修饰和折叠之间的功能联系。
