Iwaniec Brandon W J, Allegretti Madison M, Jackman Jane E
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States.
Center for RNA Biology and Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States.
Biochemistry. 2025 Aug 19;64(16):3559-3569. doi: 10.1021/acs.biochem.5c00162. Epub 2025 Aug 7.
The two-metal ion mechanism for catalysis of RNA and DNA synthesis by 5'-3' polymerases has been extensively characterized. The 3'-5' polymerase family of enzymes, consisting of tRNA guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), perform a similar nucleotide addition reaction, but in the reverse direction, adding Watson-Crick base-paired NTPs to the 5'-ends of RNA substrates. However, the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg, Mn, Co, Ni and Ca) on templated nucleotide addition activity and the kinetics of 5'-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms across diverse domains of life. This work revealed that different TLPs exhibit distinct dependencies on the concentration and identity of divalent metal ions that support effective catalysis. The patterns of metal ion usage demonstrated here for TLPs evince features that are characteristic of both canonical 5'-3' polymerases and DNA/RNA ligases. Similar to 5'-3' polymerases, some metals were also observed to be mutagenic in the context of TLP catalysis. Furthermore, we provide the first direct evidence that both ATP and the NTP poised for nucleotidyl transfer are present in the active site during the 5'-adenylylation. These results provide the first in-depth study of the role of the two-metal ion mechanism in TLP catalysis, which was initially suggested by structures of these enzymes.
5'-3'聚合酶催化RNA和DNA合成的双金属离子机制已得到广泛表征。由tRNA鸟苷酸转移酶（Thg1）和类Thg1蛋白（TLP）组成的3'-5'聚合酶家族执行类似的核苷酸添加反应，但方向相反，将沃森-克里克碱基配对的NTP添加到RNA底物的5'末端。然而，除镁之外的二价阳离子的作用尚未见报道。在此，我们研究了五种二价阳离子（镁、锰、钴、镍和钙）对模板化核苷酸添加活性以及来自生命不同领域生物的重组纯化无金属TLP催化ATP进行5'-激活动力学的影响。这项工作表明，不同的TLP对支持有效催化的二价金属离子的浓度和种类表现出不同的依赖性。这里展示的TLP使用金属离子的模式显示出典型5'-3'聚合酶和DNA/RNA连接酶所特有的特征。与5'-3'聚合酶类似，在TLP催化过程中也观察到一些金属具有诱变作用。此外，我们提供了首个直接证据，即在5'-腺苷酸化过程中，ATP和准备进行核苷酸转移的NTP都存在于活性位点。这些结果首次深入研究了双金属离子机制在TLP催化中的作用，这一作用最初是由这些酶的结构所暗示的。
