An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.
The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.
Wang
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L
Department of Bioengineering, University of Utah, Salt Lake City, UT 84108, USA.
Ruffner
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DE
D001483:Base Sequence; D001665:Binding Sites; D002712:Chlorides / Q000494:pharmacology; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D003562:Cytidine / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013891:Thiouridine / Q000737:chemistry*; D014466:Ultraviolet Rays
D002712:Chlorides; D003432:Cross-Linking Reagents; D017895:Manganese Compounds; D016337:RNA, Catalytic; D015636:Magnesium Chloride; C002908:2-thiocytidine; D013891:Thiouridine; D003562:Cytidine; D012313:RNA; C025340:manganese chloride