The global conformation of the hammerhead ribozyme determined using residual dipolar couplings.
The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.
Bondensgaard
Kent
K
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Mollova
Emilia T
ET
Pardi
Arthur
A
D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D012996:Solutions; D013816:Thermodynamics
D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012996:Solutions; C427204:hammerhead ribozyme