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. ...
Most experimental methods in structural biology provide time- and ensemble-averaged signals and, consequently, molecular structures based on such signals often exhibit only idealized, average features. Second, most experimental signals are only indirectly related to real, molecular geometries, and solving a structure typically involves a complicated procedure, which may not always result in a unique solution. To what extent do such conformationally-averaged, non-linear experimental signals and structural models derived from them accurately represent the underlying microscopic reality? Are there some structural motifs that are actually artificially more likely to be "seen" in an experiment simply due to the averaging artifact? Finally, what are the practical consequences of ignoring the averaging effects when it comes to functional and mechanistic implications that we try to glean from experimentally-based structural models? In this review, we critically address the work that has been aimed at studying such questions. We summarize the details of experimental methods typically used in structural biology (most notably nuclear magnetic resonance, X-ray crystallography and different types of spectroscopy), discuss their individual susceptibility to conformational (motional) averaging, and review several theoretical approaches, most importantly molecular dynamics simulations that are increasingly being used to aid experimentalists in interpreting structural biology experiments.
A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5 ′ exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5 ′ exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5 ′ exon and the intron. Here, we present the NMR solution structures of the d3 ′ hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg 2+ ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.
Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥ 0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.
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