An ultraviolet-inducible adenosine-adenosine cross-link reflects the catalytic structure of the Tetrahymena ribozyme.
When a shortened enzymatic version of the Tetrahymena self-splicing intervening sequence (IVS) RNA is placed under catalytic conditions and irradiated at 254 nm, a covalent cross-link forms with high efficiency. The position of the cross-link was mapped by using three independent methods: RNase H digestion, primer extension with reverse transcriptase, and partial hydrolysis of end-labeled RNA. The cross-link is chemically unusual in that it joins two adenosines, A57 and A95. Formation of this cross-link depends upon the identity and concentration of divalent cations present and upon heat-cool renaturation of the IVS in a manner that parallels conditions required for optimal catalytic activity. Furthermore, cross-linking requires the presence of sequences within the core structure, which is conserved among group I intervening sequences and necessary for catalytic activity. Together these correlations suggest that a common folded structure permits cross-linking and catalytic activity. The core can form this structure independent of the presence of P1 and elements at the 3' end of the IVS. The cross-linked RNA loses catalytic activity under destabilizing conditions, presumably due to disruption of the folded structure by the cross-link. One of the nucleotides participating in this cross-link is highly conserved (86%) within the secondary structure of group I intervening sequences. We conclude that A57 and A95 are precisely aligned in a catalytically active conformation of the RNA. A model is presented for the tertiary arrangement in the vicinity of the cross-link.
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Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
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D000241:Adenosine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000378:metabolism* / Q000528:radiation effects; D013768:Tetrahymena / Q000378:metabolism*; D014466:Ultraviolet Rays
D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D000241:Adenosine