title: Use of domain enzymes from wheat RNA ligase for in vitro preparation of RNA molecules.
abstract: Wheat RNA ligase can be dissected into three isolated domain enzymes that are responsible for its core ligase, 5'-kinase, and 2',3'-cyclic phosphate 3'-phosphodiesterase activities, respectively. In the present study, we pursued a practical strategy using the domain enzymes for in vitro step-by-step ligation of RNA molecules. As a part of it, we demonstrated that a novel side reaction on 5'-tri/diphosphate RNAs is dependent on ATP, a 2'-phosphate-3'-hydroxyl end, and the ligase domain. Mass spectroscopy and RNA cleavage analyses strongly suggested that it is an adenylylation on the 5' terminus. The ligase domain enzyme showed a high productivity for any of the possible 16 combinations of terminal bases and a high selectivity for the 5'-phosphate and 2'-phosphate-3'-hydroxyl ends. Two RNA molecules having 5'-hydroxyl and 2',3'-cyclic monophosphate groups were ligated almost stoichiometrically after separate conversion of respective terminal phosphate states into reactive ones. As the product has the same terminal state as the starting material, the next rounds of ligation are also possible in principle. Thus, we propose a flexible method for in vitro RNA ligation.
authors.affiliation: Cell-free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Makino
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Takai
mesh_terms: D001483:Base Sequence; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry; D011130:RNA Ligase (ATP) / Q000737:chemistry* / Q000302:isolation & purification; D012265:Ribonucleotides / Q000737:chemistry*; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology*
chemical_list: D012265:Ribonucleotides; D012313:RNA; D011130:RNA Ligase (ATP)