Two major tertiary folding transitions of the Tetrahymena catalytic RNA.
The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
B
B
Laggerbauer
F L
FL
Murphy
T R
TR
Cech
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent; D004492:Edetic Acid; D005296:Ferrous Compounds; D007700:Kinetics; D008274:Magnesium; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D017354:Point Mutation / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D013768:Tetrahymena / Q000235:genetics*; D014158:Transcription, Genetic
D002413:Cations, Divalent; D005296:Ferrous Compounds; D016337:RNA, Catalytic; D016053:RNA, Protozoan; C055526:Fe(II)-EDTA; D004492:Edetic Acid; D008274:Magnesium