Characterization of a native hammerhead ribozyme derived from schistosomes.
A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop-loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop-loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop-loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5' to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5' to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence.
Osborne
Edith M
EM
Department of Biochemistry, Texas A&M University, College Station, TX 77843, USA.
Schaak
Janell E
JE
Derose
Victoria J
VJ
D000818:Animals; D001483:Base Sequence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018510:RNA, Helminth / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D016337:RNA, Catalytic; D018510:RNA, Helminth; C427204:hammerhead ribozyme; D008274:Magnesium