Photocrosslinking detects a compact, active structure of the hammerhead ribozyme.
The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.
Heckman
Joyce E
JE
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Drive, 220 Stafford Hall, Burlington, Vermont 05405, USA.
Lambert
Dominic
D
Burke
John M
JM
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000378:metabolism; D010777:Photochemistry / Q000379:methods; D011743:Pyrimidines / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012550:Schistosoma mansoni / Q000201:enzymology; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D013872:Thionucleosides / Q000378:metabolism; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000378:metabolism
D003432:Cross-Linking Reagents; D009943:Organophosphorus Compounds; D011743:Pyrimidines; D016337:RNA, Catalytic; D013872:Thionucleosides; C427204:hammerhead ribozyme; C434331:phosphoramidite; D006151:Guanosine; D013891:Thiouridine; C002062:alpha-2'-deoxythioguanosine; C035169:8-bromoguanosine; C004274:4-thio-2'-deoxyuridine; D006147:Guanine; D003849:Deoxyguanosine; C030986:pyrimidine