Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

Abstract

RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5′-exon-intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg 2+ NA folding is a hierarchical process that depends on the sequential formation of secondary and tertiary structures. As the RNA phosphate-sugar backbone is negatively charged, structural compaction creates electrostatic repulsion, which must be overcome by positive charges. The majority of negative charges are nonspecifically screened by the ion atmosphere, typically a set of dynamically exchanging M + ions (1). An estimated 10-20% of negative charge is, however, compensated by M n+ that bind site-specifically to the RNA molecule, in particular, Mg 2+ (2). One RNA molecule that is known to harbor several specific M 2+ binding sites is the self-splicing group II intron Sc.ai5γ from the yeast mitochondrial cox1 (cytochrome oxidase 1) gene (3). It is one of the largest known RNA enzymes, and both its folding pathway and catalysis are strictly dependent on Mg 2+. In turn, the splicing reaction is inhibited by small amounts of Ca 2+ (4). Site specificity of the two sequential transesterfication reactions is ensured by proper base pairing between distal exon-binding sites (5′ cleavage, EBS1 and 2; 3′ cleavage, EBS3) and intron-binding sites (IBS1, 2, and 3) (5).Single-molecule Förster resonance energy transfer (smFRET), i.e., distance-dependent energy transfer between a single pair of fluorophores, is ideally suited to study the cation-dependent conformational dynamics of single RNA molecules (6, 7). If different conformations lead to distinctly different transfer efficiencies, smFRET unveils the entire folding pathway, reports on the relative occurrence of all conformations present in the ensemble, and provides detailed information on the rates at which they interconvert (7). This is important because simple two-state folding is rarely observed in experimental data (8, 9). Rather, the vast conformational space sampled by biomolecules often results not only in folding intermediates but also in kinetic traps and/or multiple native states. In an smFRET experiment, individual molecules consequently display different behaviors that may or may not persist over the observation period (10). Heterogeneity has been precedented for a number of RNA molecules, including group I introns (11, 12), the hairpin ribozyme (13-17), and RNase P RNA (18). In addition, heterogeneity has been reported for different . However, the molecular basis of the phenomenon is often enigmatic, and its quantitative characterization is challenging (21).Here we use the 5′-exon-intron recognition site of the Sc.ai5γ ribozyme to study Mg 2+ -and Ca 2+ -mediated RNA-RNA structure formation by smFRET. ...

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

“…4b). Diffuse binding is expected to be similar for RNA and DNA exons, thus the shift of the midpoint of the binding curve (RNA-RNA: [Mg 2+ ] mid = 3.7 ± 0.9 mM versus RNA-DNA: [Mg 2+ ] mid = 7.9 ± 1.9 mM) suggests a tighter innerand/or outer-sphere coordination of Mg 2+ at the RNA-RNA interface in line with previous binding affinities calculated from NMR chemical shifts 15,40 .…”

“…Like a padlock, the metal ion packs the strands together, thereby retaining the exon in the active site for a longer time than if no gatekeeping ion was present. As ion coordination is transient though, designated binding pockets are often only partially occupied and exon dissociation is thus kinetically heterogeneous 15 . Particularly, Mg 2+ 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] .…”

“…Single-molecule techniques are well suited to detect subpopulations of molecules in different conformational states 15,[19][20][21] . This involves ion-induced collapse of RNA secondary structure elements as well as formation of more distant tertiary contacts 17 .…”

Decoding Single Molecule Time Traces with Dynamic Disorder

“…It is not easy to elucidate the molecular origin of disorder in a conclusive manner; yet, it has recently been suspected that interactions of biomolecules with cofactor such as ATP and multivalent metal-ions could be the microscopic causes for those molecules exhibiting dynamical heterogeneity [12,13,16,88,89]. Modulating the concentration of Mg 2+ ions from high to low and again to high induced inter-conversions of dynamic patterns in equilibrium conformational fluctuations of T. ribozyme [12] and Holliday junctions [13].…”

Physics‐based all‐atom modeling of RNA energetics and structure

“…Parameterizations of these ions are fraught even in the context of polarizable force fields . For example, the lifetime of RNA‐divalent cation interactions can be on the order of milliseconds, possibly longer . This is longer than most MD simulations.…”