Multiple folded conformations of a hammerhead ribozyme domain under cleavage conditions.
The conformation of a hammerhead ribozyme domain, formed between a 35-mer ribozyme and its 14-mer substrate, was studied under cleavage conditions with non-cleavable substrate analogues. Each analogue substrate contained a single 2'-deoxy-4-thiouridine that formed specific intermolecular crosslinks within the ribozyme-substrate complex upon irradiation with 365 nm light. The residues at positions 7 and 8 to 9 (the cleavage site) were found in contact with several bases of the ribozyme 5' conserved region regardless of whether the substrate was all-RNA, with a single deoxynucleotide at the cleavage site, or all-DNA. These contacts were observed in the presence of either 20 mM Mg2+ or 200 mM Na+. The multiple crosslinks generated between the ribozyme central core and each of the three substrates suggest the existence of several folded conformers of the ribozyme. A ribozyme mutation (A28U), which abolishes the catalytic activity, was shown to strongly affect the pattern of crosslinks; this argues for the presence of multiple folded conformers of the ribozyme some of which may be catalytically inactive.
Woisard
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Institut Jacques Monod, CNRS-Université Paris 7, France.
Fourrey
J L
JL
Favre
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D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives; D006678:HIV / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013891:Thiouridine / Q000031:analogs & derivatives; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009692:Nucleic Acid Heteroduplexes; D016337:RNA, Catalytic; D012367:RNA, Viral; C070455:2'-deoxy-4'-thiouridine; D013891:Thiouridine; D004247:DNA; D003857:Deoxyuridine