A kinetic intermediate that regulates proper folding of a group II intron RNA.
The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified. Employing time-resolved nucleotide analog interference mapping, we have identified a cluster of atoms within the D1 main stem that control the rate constant for D135 collapse. Functional groups within the kappa-zeta element are particularly important for this earliest stage of folding, which is intriguing given that this same motif also serves later as the docking site for catalytic domain 5. More important, the kappa-zeta element is shown to be a divalent ion binding pocket, indicating that this region is a Mg(2+)-dependent switch that initiates the cascade of D135 folding events. By measuring the Mg(2+) dependence of the compaction rate constant, we conclude that the actual rate-limiting step in D1 compaction involves the formation of an unstable folding intermediate that is captured by the binding of Mg(2+). This carefully orchestrated folding pathway, in which formation of an active-site docking region is early and rate limiting, ensures proper folding of the intron core and faithful splicing. It may represent an important paradigm for the folding of large, multidomain RNA molecules.
Waldsich
Christina
C
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.
Pyle
Anna Marie
AM
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044563:RNA, Ribosomal, Self-Splicing / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
C506264:D135 ribozyme, S cerevisiae; D016337:RNA, Catalytic; D012331:RNA, Fungal; D044563:RNA, Ribosomal, Self-Splicing; D012313:RNA; D008274:Magnesium