Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes.
Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid-base catalysis, and are >15 A from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.
Lambert
Dominic
D
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Drive, 220B Stafford Hall, Burlington, Vermont 05405, USA.
Heckman
Joyce E
JE
Burke
John M
JM
D001483:Base Sequence; D006151:Guanosine / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D014466:Ultraviolet Rays
D016337:RNA, Catalytic; D006151:Guanosine