Time-resolved Hydroxyl Radical Footprinting Research Articles

Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.

The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The ‘fast’, ‘intermediate’ and ‘slow’ H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all ‘fast’ protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.

Assembly of the 30s Ribosome From the RNA Folding Perspective

Ribosome assembly requires folding of the rRNA and the hierarchical addition of 20 or more proteins to the complex. We visualized assembly of the bacterial 30S ribosomal subunit in real time using time-resolved hydroxyl radical footprinting. This method reveals the extent of RNA and protein interactions at each segment of the RNA backbone, providing a detailed view of the changes to the rRNA structure during assembly. Each domain of the 30S ribosome assembles concurrently in vitro, and many tertiary RNA interactions and RNA-protein interactions are established within the first 0.1 seconds. Individual proteins protect different segments of their binding site at different rates, suggesting that the initial protein-RNA complexes are remodeled during assembly. By perturbing the free energy of RNA-protein complexes from the body of the 30S subunit, we find that a single protein can stabilize an entire domain of the 16S rRNA. However, multiple proteins bound to the same domain narrow the ensemble of rRNA conformations. Specific structural switches stabilize the decoding active site and enable long-range structural communication within the 30S ribosomal subunit.

A Rate-Limiting Conformational Step in the Catalytic Pathway of the glmS Ribozyme

The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the downregulation of glmS gene expression. Unlike other riboswitches, the glmS ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand. This observation led us to test if individual steps in the reaction pathway could be dissected by initiating the cleavage reaction before or after Mg(2+)-dependent folding. Here we show that self-cleavage reactions initiated with simultaneous addition of Mg(2+) and GlcN6P are slow (3 min(-1)) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg(2+)-containing buffer (72 min(-1)). These data indicate that some level of Mg(2+)-dependent folding is rate-limiting for catalysis. Reactions initiated by addition of GlcN6P to the prefolded ribozyme also resulted in a 30-fold increase in the apparent ligand K(d) compared to those of reactions initiated by a global folding step. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. The results of these experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13 min(-1)). This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap.

Complementing global measures of RNA folding with local reports of backbone solvent accessibility by time resolved hydroxyl radical footprinting

A variety of analytical techniques are used to probe the mechanisms by which RNA molecules fold to discrete three dimensional structures. Methods such as small angle X-ray scattering (SAXS) report global properties like overall size and shape of the RNA. Other methods such as chemical or enzymatic mapping (footprinting) report properties with resolution as fine as single nucleotide. The hydroxyl radical (•OH) is a footprinting probe which cleaves the oligonucleotide backbone independently of sequence and thus is a valuable reporter of backbone solvent accessibility. Combinations of global and local measures of folding reactions are uniquely able to distinguish specific from nonspecific processes. This article highlights the application of •OH footprinting as a complement to SAXS for kinetics analysis of RNA folding. We illustrate this combination of techniques using a study of the role played by the stiffness of a hinge in determining the rate limiting step of a Mg 2+-mediated RNA folding reaction.

Identifying Energy Barriers in RNA Folding Through Kinetic Model Enumeration

Large RNA molecules are known to fold through multiple parallel pathways to achieve their functional conformation. These pathways include stable intermediate structures that we identify using our KinFold algorithm (http://simtk.org/home/kinfold) for the analysis of time-resolved hydroxyl radical footprinting experiments. KinFold enumerates kinetic model topologies to determine the structure, lifetimes, and abundance of intermediates along the folding pathways of RNA. We are therefore able to identify the best fitting kinetic model for each folding reaction studied. We used this approach to study the folding of the L-21 T. thermophila group I intron, a catalytic ribozyme, at 6 temperatures ranging from 25° to 51° C. Our analysis of this data reveals that the rates for a majority of the transitions (between intermediate, folded and unfolded states) obey the Arrhenius equation, allowing us to estimate the relative energies of activation for inter conversion between the states of an RNA. Furthermore, we are able to estimate the lifetimes of the different intermediate structures. These results reveal a simple landscape, where folding of the peripheral elements of the RNA create significant barriers to folding. Our approach is unique in that it allows us to simultaneously estimate the activation energies for all transitions in an RNA folding reaction.

Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.

Tertiary Interactions Determine the Accuracy of RNA Folding

RNAs must fold into unique three-dimensional structures to function in the cell, but how each polynucleotide finds its native structure is not understood. To investigate whether the stability of the tertiary structure determines the speed and accuracy of RNA folding, docking of a tetraloop with its receptor in a bacterial group I ribozyme was perturbed by site-directed mutagenesis. Disruption of the tetraloop or its receptor destabilizes tertiary interactions throughout the ribozyme by 2-3 kcal/mol, demonstrating that tertiary interactions form cooperatively in the transition from a native-like intermediate to the native state. Nondenaturing PAGE and RNase T1 digestion showed that base pairs form less homogeneously in the mutant RNAs during the transition from the unfolded state to the intermediate. Thus, tertiary interactions between helices bias the ensemble of secondary structures toward native-like conformations. Time-resolved hydroxyl radical footprinting showed that the wild-type ribozyme folds completely within 5-20 ms. By contrast, only 40-60% of a tetraloop mutant ribozyme folds in 30-40 ms, with the remainder folding in 30-200 s via nonnative intermediates. Therefore, destabilization of tetraloop-receptor docking introduces an alternate folding pathway in the otherwise smooth energy landscape of the wild-type ribozyme. Our results show that stable tertiary structure increases the flux through folding pathways that lead directly and rapidly to the native structure.

Communication Between RNA Folding Domains Revealed by Folding of Circularly Permuted Ribozymes

To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1–3 min at 42 °C. Permutation of the 5′ end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 °C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5–8 min). By contrast, circular permutation of the 5′ end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60–80% folded within 30 s. RNase T 1 digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5′-to-3′ order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme

The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant E(deltaP5abc) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E(deltaP5abc) forms its earliest stable tertiary structure on the millisecond time scale, approximately 5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E(deltaP5abc) in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E(deltaP5abc) ribozyme is much slower (approximately 0.6 min(-1)), in a manner similar to that of the wild type. Also similar, only a small fraction of E(deltaP5abc) attains the native state on this time scale under standard conditions at 25 degrees C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E(deltaP5abc) refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.

Communication between RNA folding domains revealed by the tertiary folding pathway of circularly permuted ribozymes

RNA folding is governed by a balance of long-range and short-range interactions. Circular permutation changes the distance between interacting residues in the primary sequence, without significantly changing the native fold of the RNA. To study the role of topology in RNA folding, we determined the equilibrium and kinetic folding pathways of two circularly permuted variants of the Tetrahymena rRNA group I intron, using time-resolved hydroxyl radical footprinting and oligonucleotide probes. Permutation of the 5′ end to G111 in the P4 helix allowed the P4-P6 domain to fold five times more rapidly than it does in the wild type RNA; the remainder of the intron folded five times more slowly. By contrast, permutation of the 5′ end to G303 in J8/7 allowed peripheral tertiary interactions to form more rapidly than in the wild type RNA intron but slowed the acquisition of native tertiary interactions in the catalytic center. RNase T1 digestion and FMN photocleavage structure mapping suggest that circular permutation of the intron sequence alters the initial ensemble of secondary structures, thereby changing the structure of the tertiary folding intermediates. Discontinuities in the RNA backbone disrupt folding communication between structural domains, perturbing and delaying organization of the catalytic center, implying that the 5′-to-3′ order of domains are subject to selective pressure during evolution. Supported by NIH GM60809 (S.W.)

Productive folding to the native state by a group II intron ribozyme

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5γ. The isotherms span a similar range of Mg 2+ concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5γ intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5γ occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.