Productive folding to the native state by a group II intron ribozyme.
Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.
Swisher
Jennifer F
JF
Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, NY 10032, USA.
Su
Linhui J
LJ
Brenowitz
Michael
M
Anderson
Vernon E
VE
Pyle
Anna Marie
AM
D020029:Base Pairing / Q000187:drug effects; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D017665:Hydroxyl Radical / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012313:RNA / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D000077278:RNA, Mitochondrial; D017186:Titrimetry; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
D016337:RNA, Catalytic; D012331:RNA, Fungal; D000077278:RNA, Mitochondrial; D017665:Hydroxyl Radical; D012313:RNA; D008274:Magnesium