Group II intron folding under near-physiological conditions: collapsing to the near-native state.
The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 degrees C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions. We observe that compaction of the RNA proceeds slowly to completion, even at low magnesium concentration (3 mM). Kinetic analysis shows that this compact species is a "near-native" intermediate state that is readily chased into the native state by the addition of high salt. Structural probing reveals that the near-native state represents a compact domain 1 scaffold that is not yet docked with the catalytic domains (D3 and D5). Interestingly, native ribozyme reverts to the near-native state upon reduction in magnesium concentration. Therefore, while the intron can sustain the intermediate state under physiological conditions, the native structure is not maintained and is likely to require stabilization by protein cofactors in vivo.
Fedorova
Olga
O
Howard Hughes Medical Institute Yale University, New Haven, CT 06520, USA.
Waldsich
Christina
C
Pyle
Anna Marie
AM
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D007438:Introns; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D044563:RNA, Ribosomal, Self-Splicing / Q000737:chemistry*; D013463:Sulfuric Acid Esters / Q000737:chemistry; D013768:Tetrahymena / Q000737:chemistry / Q000235:genetics*; D013816:Thermodynamics
D016337:RNA, Catalytic; D044563:RNA, Ribosomal, Self-Splicing; D013463:Sulfuric Acid Esters; D008274:Magnesium; C007482:dimethyl sulfate