Hairpin ribozymes with four-way helical junctions mediate intracellular RNA ligation.
Virtually all RNA-mediated reactions require transitions among alternative RNA conformations. The complexity of biological reactions can obscure specific conformational changes in vivo and important features of the intracellular environment are difficult to reproduce in vitro. However, simple RNA self-cleavage and ligation reactions offer a unique opportunity to measure the kinetics and equilibria of specific RNA conformational transitions directly in living cells. Hairpin ribozymes that incorporate the natural four-way helical junction self-cleave rapidly in vivo, but only when cleavage products dissociate rapidly. Cleavage rates fall when cleavage products remain bound in stable base-paired helices, providing evidence that bound products undergo re-ligation. These results provide the first detailed kinetic description of an intracellular ribozyme reaction that includes cleavage, ligation and product dissociation rates. Kinetic and equilibrium parameters measured in vivo correspond well, but not perfectly, with values measured for the same reactions in vitro under conditions that approximate an intracellular ionic environment.
Yadava
R S
RS
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute MB35, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Choi
A J
AJ
Lebruska
L L
LL
Fedor
M J
MJ
D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009994:Osmolar Concentration; D020871:RNA Stability / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics
D002412:Cations; D016337:RNA, Catalytic; D015636:Magnesium Chloride; D012965:Sodium Chloride