Abstract
The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.
Highlights
The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons
The two intensity signals are converted into transfer efficiencies that fluctuate between a zero Förster resonance energy transfer (FRET) state, corresponding to the unbound hairpin, and a high FRET state around 0.75 of the formed tertiary contact
Static traces in the high FRET state only appear in the presence of intron binding site 1 (IBS1)*, but not dIBS1* (Fig. 1e)
Summary
The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. The long-range tertiary interactions embed the 5′-exon in the active core of the ribozyme (Fig. 1b)[6,7,8], where a hydrogen-bond network and several coordinating metal ions convey stability to the tertiary contacts[9,10]. If the association of intron and exon is too strong, the ribozyme no longer discriminates between correct and mismatched targets, which may lead to gene disruption and disease if reverse splicing occurs in tumor suppressor genes like p5312,13. To minimize such errors, most group II introns use two independent exon recognition sites to keep hold of the 5′-exon: exon binding site 1. Mg2+ is known to induce such kinetic partitioning by interacting with RNA directly (inner-sphere coordination) or via a water molecule (outer-sphere coordination)[16,17,18]
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