Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme

Abstract

Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from Azoarcus pre-tRNA ile . Base pairing of the ribozyme core requires 10-fold less Mg 2؉ than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37°C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the Azoarcus ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4 -P6 and P3-P9 domains, that may explain the unusual stability of the Azoarcus ribozyme and the cooperativity of folding.RNA modeling ͉ RNA structure ͉ metal ions ͉ hydroxyl radical footprinting T he assembly of RNA into functional structures underlies many steps in gene expression and regulation. Recent work has outlined the Mg 2ϩ -dependent folding pathways of large ribozymes (1, 2). Experimental and theoretical results suggest that the initial association of divalent cations induces collapse of the extended RNA chain into more compact structures that favor formation of tertiary interactions (3-8). The structure of the compact intermediates and the extent to which these interactions lead to the native conformation are not yet characterized.On the one hand, individual domains of the Tetrahymena group I ribozyme and the catalytic domain of RNase P fold on a time scale (10-100 ms) similar to the initial collapse transitions of the ribozyme (6, 9, 10). On the other hand, nonspecific collapse results in an ensemble of native and non-native conformations. Many larger RNAs, such as the Tetrahymena group I and Bacillus subtilis RNase P ribozymes, fold slowly in vitro because a large fraction of the RNA population becomes trapped in misfolded intermediates (11,12). An important question is whether RNAs with a simpler 3D architecture are more likely to fold directly to the native structure, as expected from theoretical models (4). Furthermore, we want to know whether the likelihood of misfolding can be predicted from the specificity of counterion-induced collapse.We investigated the Mg 2ϩ -dependent equilibrium folding pathway and constructed a 3D model of the Azoarcus group I intron. The 205-nt group IC3 intron in the pre-tRNA ile of the Azoarcus bacterium is the smallest known self-splicing group I intron (13). It retains the conserved catalytic core common to all group I introns, but lacks the peripheral domains that stabilize folding intermediates of the larger Tetrahymena intron (14).We observed two macroscopic folding transitions in the Azoarcus ribozyme...

Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA

“…In 20 mM MgCl 2 , 10% of the pre-tRNA folded in the deadtime of the assay (, 30 s), and the remainder reached the native state at a rate of 0.47 min 21 ( Figure 4A). Thus, the average folding time of the pre-tRNA is much longer than that of the ribozyme, 9 consistent with the presence of metastable intermediates.…”

“…We have found that the Azoarcus L-9 ribozyme folds in 50 -100 ms in a concerted fashion. 9 Here, we find that correct folding of the pre-tRNA is delayed by non-native base-pairs between the tRNA and the intron. Thus, the exons contribute to splice site recognition, but increase the probability of misfolding in vitro.…”

“…9 At first glance, the pre-tRNA appeared to follow a similar pathway, judging from changes in the extent of RNase T 1 and hydroxyl radical protection with increasing concentration of MgCl 2 . Much of the pre-tRNA secondary structure is formed in 10 mM Tris -HCl (pH 7.5) or Tris -HCl plus 20 mM MgCl 2 (blue arrowheads in Figure 1).…”

“…18 Because the Azoarcus L-9 ribozyme folds rapidly ðt ¼ 50 msÞ, 40% of the ribozyme RNA reaches the native state (N) during the brief time that the sample encounters Mg 2þ in the gel running buffer. 9 The native RNA appears as a sharp band in the gel, and further refolding is largely arrested after the RNA enters the gel matrix. By contrast, only 10% of the pre-tRNA folded correctly in the deadtime of the native gel assay ( Figure 3B), when the RNA was not pre-incubated in buffer containing Mg 2þ .…”

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

“…44 Solvent accessibility of the IRES was probed using Fe(II)-EDTA and H 2 O 2 in a solution of RNA renatured in the presence or absence of IRAB (20 mM) followed by primer extension analysis with fluorescently labeled primers, resolved by capillary electrophoresis. The results yielded cleavage information at single nucleotide resolution, reporting on solvent exposure of the IRES nucleotides (Fig.…”