Jiang Hengyi, Liu Getong, Gao Yanqing, Gan Jianhua, Chen Dongrong, Murchie Alastair I H
Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China and Institutes of Biomedical Sciences, Shanghai Medical College, Key Laboratory of Medical Epigenetics and Metabolism, Fudan University, Shanghai, China; Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Department of Physiology and Biophysics, Shanghai Public Health Clinical Center, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, China.
J Biol Chem. 2024 Nov;300(11):107863. doi: 10.1016/j.jbc.2024.107863. Epub 2024 Oct 5.
The methyltransferase ribozyme SMRZ-1 utilizes S-adenosyl-methionine (SAM) and Cu (II) ions to methylate RNA. A comparison of the SAM-bound and unbound RNA structures has shown a conformational change in the RNA. However, the contribution of specific interactions and the role of a pseudo-triplex motif in the catalytic center on the methylation reaction is not completely understood. In this study, we have used atomic substitutions and mutational analysis to investigate the reaction specificity and the key interactions required for catalysis. Substitution of the fluorescent nucleotide 2-aminopurine within the active ribozyme enabled the conformational dynamics of the RNA upon co-factor binding to be explored using fluorescence spectroscopy. We show that fast co-factor binding (t ∼ 0.7 s) drives a conformational change in the RNA to facilitate methyl group transfer. The importance of stacking interactions at the pseudo-triplex motif and chelation of the Cu (II) ion were shown to be essential for SAM binding.
甲基转移酶核酶SMRZ-1利用S-腺苷甲硫氨酸（SAM）和铜（II）离子对RNA进行甲基化。对结合SAM和未结合SAM的RNA结构进行比较，结果显示RNA发生了构象变化。然而，特定相互作用的贡献以及催化中心中假三链体基序在甲基化反应中的作用尚未完全明确。在本研究中，我们通过原子取代和突变分析来研究反应特异性以及催化所需的关键相互作用。在活性核酶中取代荧光核苷酸2-氨基嘌呤，使得能够利用荧光光谱法探索辅因子结合后RNA的构象动力学。我们发现快速的辅因子结合（t ∼ 0.7 s）会驱动RNA发生构象变化，以促进甲基转移。结果表明，假三链体基序处的堆积相互作用以及铜（II）离子的螯合对于SAM结合至关重要。
