Fast cleavage kinetics of a natural hammerhead ribozyme.
The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.
Canny
Marella D
MD
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309-0215, USA.
Jucker
Fiona M
FM
Kellogg
Elizabeth
E
Khvorova
Anastasia
A
Jayasena
Sumedha D
SD
Pardi
Arthur
A
D000818:Animals; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012547:Schistosoma / Q000201:enzymology / Q000235:genetics
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008274:Magnesium