Hamachi Kokoro, Mutsuro-Aoki Hiromi, Tanizawa Kana, Hirasawa Ito, Umehara Takuya, Tamura Koji
Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.
Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Biosystems. 2019 Mar;177:9-15. doi: 10.1016/j.biosystems.2019.01.004. Epub 2019 Jan 11.
The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, and <hairpin-ΔU>, the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in <hairpin-ΔU>, but not in , despite the absence of a substrate-binding site in <hairpin-ΔU>. Loop-loop interactions of - and <hairpin-ΔU>-variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.
通过引入互补的7元环形成接吻环，已知可显著提高截短的R3C连接酶核酶的活性，否则其活性会降低。活性的恢复被认为是由于接吻复合物形成诱导的具有互补环的两个分子的重排。通过组合两种类型的R3C连接酶核酶突变体和<发夹-ΔU>，研究了环组成对连接活性的影响。底物连接发生在<发夹-ΔU>中，但在中不发生，尽管<发夹-ΔU>中没有底物结合位点。变体和<发夹-ΔU>变体与互补6元环的环-环相互作用也导致了适当的接吻复合物形成诱导的底物连接。然而，7元环和6元环以及/或者6元环和5元环的异质组合产生了不同的结果，这取决于环区域的序列和凸起核苷酸。这些差异表明，热力学和动力学控制都作用于接吻环相互作用介导的缩短的反式R3C核酶的重排。
