title: Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants.
abstract: The catalytic properties of the hammerhead ribozyme embedded in the (+) strand of the satellite tobacco ringspot viral genome are analyzed with the goal of obtaining the elemental rate constants of the cleavage (k(2)) and ligation (k(-)(2)) steps. Two different chimeras combining the sTRSV (+) hammerhead and the well-characterized hammerhead 16 were used to measure the cleavage rate constant (k(2)), the rate of approach to equilibrium (k(obs) = k(2) + k(-)(2)), and the fraction of full-length hammerhead at equilibrium (k(-)(2)/k(2) + k(-)(2)). When compared to minimal hammerheads that lack the recently discovered loop I-loop II interaction, an extended format hammerhead derived from sTRSV studied here shows at least a 20-fold faster k(2) and a 1300-fold faster k(-)(2) at 10 mM MgCl(2). However, the magnesium dependence of the cleavage rate is not significantly changed. Thus, the enhanced cleavage of this hammerhead observed in vivo is due to its higher intrinsic rate and not due to its tighter binding of magnesium ions. The faster k(-)(2) of this hammerhead suggests that ligation may be used to form circular RNA genomes. This in vitro system will be valuable for experiments directed at understanding the hammerhead mechanism and the role of the loop I-loop II interaction.
authors.affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Hogan 2-100, Evanston, Illinois 60208, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Nelson
authors.forename: Irina
authors.initials: I
authors.lastname: Shepotinovskaya
authors.forename: Olke C
authors.initials: OC
authors.lastname: Uhlenbeck
mesh_terms: D000818:Animals; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral; D019400:Tobacco mosaic satellite virus / Q000201:enzymology*
chemical_list: D016337:RNA, Catalytic; D012367:RNA, Viral; C427204:hammerhead ribozyme