Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis.
Tertiary folding of the hammerhead ribozyme has been analyzed by hydroxyl radical footprinting. Three hammerhead constructs with distinct noncore sequences, connectivities, and catalytic properties show identical protection patterns, in which conserved core residues (G5, A6, U7, G8, and A9) and the cleavage site (C17, G1.1, and U1.2) become reproducibly protected from nucleolytic attack by radicals. Metal ion titrations show that all protections appear together, suggesting a single folding event to a common tertiary structure, rather than an ensemble of different folds. The apparent binding constants for folding and catalysis by Mg(2+) are lower than those for Li(+) by 3 orders of magnitude, but in each case the protected sites are identical. For both Mg(2+) and Li(+), the ribozyme folds into the protected tertiary structure at significantly lower cation concentrations than those required for cleavage. The sites of protection include all of the sites of reduced solvent accessibility calculated from two different crystal structures, including both core and noncore nucleotides. In addition, experimentally observed protected sites include additional sequences adjacent to those predicted by the crystal structures, suggesting that the solution structure may be folded into a more compact shape. A 2'-deoxy substitution at G5 abolishes all protection, indicating that the 2'-OH is essential for folding. Together, these results support a model in which low concentrations of metal ions fold the ribozyme into a stable ground state tertiary structure that is similar to the crystallographic structures, and higher concentrations of metal ions support a transient conformational change into the transition state for catalysis. These data do not themselves address the issue as to whether a large- or small-scale conformational change is required for catalysis.
Hampel
Ken J
KJ
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, The University of Vermont, Burlington, Vermont 05405, USA.
Burke
John M
JM
D008274:Magnesium / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents / Q000378:metabolism
D016337:RNA, Catalytic; D012997:Solvents; C427204:hammerhead ribozyme; D008274:Magnesium