Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
RNA. 2024 Sep 16;30(10):1306-1314. doi: 10.1261/rna.080120.124.
Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO and 5'-PO RNA ends to form a 2'-PO,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO RNA terminus to form an RNA-adenylate intermediate (AppRNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNAp substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.
真菌 RNA 连接酶（LIG）是一种必需的 tRNA 剪接酶，它将 3'-OH、2'-PO 和 5'-PO RNA 末端连接起来，形成 2'-PO、3'-5'磷酸二酯键剪接连接点。连接需要三个依赖二价阳离子的腺苷转移步骤。首先，LIG 与 ATP 反应形成共价连接酶-（赖氨酸-Nζ）-AMP 中间物并取代焦磷酸。其次，LIG 将 AMP 转移到 5'-PO RNA 末端，形成 RNA-腺苷酸中间物（AppRNA）。第三，LIG 指导 RNA 3'-OH 攻击 AppRNA 以形成剪接连接点并取代 AMP。真菌 LIG 与典型的多核苷酸连接酶的区别特征是需要 2'-PO 来合成 3'-5'磷酸二酯键。真菌 LIG 由一个 N 端腺苷转移酶结构域和一个独特的 C 端结构域组成。LIG 的 C 端结构域（CthLIG）与一个硫酸盐阴离子结合，该阴离子被认为是末端 2'-PO 的模拟物。在这里，我们研究了 C 结构域和保守的硫酸盐配体（His227、Arg334、Arg337）对 pRNAp 底物连接的贡献。我们发现 C 结构域对于末端连接是必需的，但对于连接酶腺苷酸化是可有可无的。H227A、R334A 和 R337A 突变分别使第二步 RNA 腺苷酸化的速率减慢了 420 倍、120 倍和 60 倍，而野生型 CthLIG 的速率减慢了 580 倍。R334A-R337A 双突变使第二步反应的速度减慢了 580 倍。这些结果有力地证明了严格保守的 His-Arg-Arg 三肽是真菌 tRNA 连接酶的 2'-PO 末端特异性的执行者，也是真菌 tRNA 剪接小分子干预的靶点。
