Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Dynamics of biological macromolecules: not a simple slaving by hydration water.

We studied the dynamics of hydrated tRNA using neutron and dielectric spectroscopy techniques. A comparison of our results with earlier data reveals that the dynamics of hydrated tRNA is slower and varies more strongly with temperature than the dynamics of hydrated proteins. At the same time, tRNA appears to have faster dynamics than DNA. We demonstrate that a similar difference appears in the dynamics of hydration water for these biomolecules. The results and analysis contradict the traditional view of slaved dynamics, which assumes that the dynamics of biological macromolecules just follows the dynamics of hydration water. Our results demonstrate that the dynamics of biological macromolecules and their hydration water depends strongly on the chemical and three-dimensional structures of the biomolecules. We conclude that the whole concept of slaving dynamics should be reconsidered, and that the mutual influence of biomolecules and their hydration water must be taken into account.

Dynamics of tRNA at different levels of hydration.

The influence of hydration on the nanosecond timescale dynamics of tRNA is investigated using neutron scattering spectroscopy. Unlike protein dynamics, the dynamics of tRNA is not affected by methyl group rotation. This allows for a simpler analysis of the influence of hydration on the conformational motions in RNA. We find that hydration affects the dynamics of tRNA significantly more than that of lysozyme. Both the characteristic length scale and the timescale of the conformational motions in tRNA depend strongly on hydration. Even the characteristic temperature of the so-called "dynamical transition" appears to be hydration-dependent in tRNA. The amplitude of the conformational motions in fully hydrated tRNA is almost twice as large as in hydrated lysozyme. We ascribe these differences to a more open and flexible structure of hydrated RNA, and to a larger fraction and different nature of hydrophilic sites. The latter leads to a higher density of water that makes the biomolecule more flexible. All-atom molecular-dynamics simulations are used to show that the extent of hydration is greater in tRNA than in lysozyme. We propose that water acts as a "lubricant" in facilitating enhanced motion in solvated RNA molecules.

RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.

Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Macromolecular complexes: how RNA and protein get together.

Recent studies of protein-dependent RNA splicing have provided new insights into the way RNA-protein interactions drive the stepwise assembly of catalytic RNA structures.

An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence.

A highly conserved ribosomal stem-loop immediately upstream of the Tetrahymena splice junction can inhibit both forward and reverse self-splicing by competing with base pairing between the 5' exon and the guide sequence of the intervening sequence. Formation of this unproductive hairpin is preferred in precursor RNAs with short exons and results in a lower rate of splicing. Inhibition of self-splicing is not observed in longer precursors, suggesting that additional interactions in the extended exons can influence the equilibrium between the productive and unproductive hairpins at the 5' splice site. An alternative pairing upstream of the 5' splice site has been identified and is proposed to stabilize the active conformer of the pre-rRNA. Nucleotide changes that alter the ability to form this additional helix were made, and the self-splicing rates were compared. Precursors in which the proposed stem is stabilized splice more rapidly than the wild type, whereas RNAs that contain a base mismatch splice more slowly. The ability of DNA oligomers to bind the RNA, as detected by RNase H digestion, correlates with the predicted secondary structure of the RNA. We also show that a 236-nucleotide RNA containing the natural splice junction is a substrate for intervening sequence integration. As in the forward reaction, reverse splicing is enhanced in ligated exon substrates in which the alternative rRNA pairing is more stable.

Structure and dynamics of ribosomal RNA.

Over the past two years, progress in X-ray crystallography, NMR spectroscopy and electron microscopy has begun to reveal the complex structure of the RNA within the ribosome. The structures of ribosomal proteins L11 and S15, among others, show how RNA-protein interactions organize the conformation of the junctions between ribosomal RNA helices. Genetic and biochemical methods have also identified a three base-pair switch within the 16S rRNA that is linked to mRNA decoding.

Folding intermediates of a self-splicing RNA: mispairing of the catalytic core.

The Tetrahymena thermophila self-splicing RNA is trapped in an inactive conformation during folding reactions at physiological temperatures. The structure of this metastable intermediate was probed by chemical modification interference and site-directed mutagenesis. In the inactive structure, an incorrect base-pairing, which we call Alt P3, displaces the P3 helix in the catalytic core of the intron. Mutations that stabilize Alt P3 increase the fraction of pre-rRNA that becomes trapped in the inactive structure, whereas mutations that destabilize Alt P3 reduce accumulation of this conformer. At high concentrations of Mg2+, the yield of correctly folded mutant pre-rRNAs is similar to wild-type RNA. Under these conditions, the rate of folding for mutant RNAs is slower than for the wild-type, but is increased by addition of urea. The results show that slow folding of the Tetrahymena pre-rRNA is a consequence of non-native secondary structure in the catalytic core of the intron, which is linked to an alternative hairpin in the 5' exon. This illustrates how kinetically stable, long-range interactions shape RNA folding pathways.

Facilitation of group I splicing in vivo: misfolding of the Tetrahymena IVS and the role of ribosomal RNA exons.

Self-splicing of the group I IVS from Tetrahymena thermophila rDNA is limited by the time required for the RNA to reach its active conformation. In vitro, folding is slow because the pre-rRNA becomes kinetically trapped in inactive structures. In vivo, splicing is 50 times more rapid, implying that misfolding of the pre-rRNA is corrected. Exon mutations that inhibit self-splicing 100-fold in vitro were fully rescued when the pre-rRNA containing the IVS was expressed in E. coli. In contrast, IVS mutations that cause misfolding were only partially suppressed at 42 degrees C, and doubled the activation energy of splicing. These results suggest that intracellular folding of the pre-rRNA involves metastable intermediates similar to those observed in vitro. Precursors with natural rRNA exons were more active and less cold-sensitive than those with non-rRNA exons. This shows that the rRNA reduces misfolding of the IVS, thereby facilitating splicing of the pre-rRNA in vivo.

The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA.

Bas?e-pairing between the terminal loops of helices P2.1 and P9.1a (P13) and P2 and P5c (P14) stabilize the folded structure of the Tetrahymena group I intron. Using native gel electrophoresis to analyze the folding kinetics of a natural pre-RNA containing the Tetrahymena intron, we show that P13 and P14 are the only native loop-loop interactions among six possible combinations. Other base-pairing interactions of the loop sequences stabilize misfolded and inactive pre-RNAs. Mismatches in P13 or P14 raised the midpoints and decreased the cooperativity of the Mg(2+)-dependent eqXuilibrium folding transitions. Although some mutations in P13 resulted in slightly higher folding rates, others led to slower folding compared to the wild-type, suggesting that P13 promotes formation of P3 and P7. In contrast, mismatches in P14 increased the rate of folding, suggesting that base-pairing between P5c and P2 stabilizes intermediates in which the catalytic core is misfolded. Although the peripheral helices stabilize the native structure of the catalytic core, our results show that formation of long-range interactions, and competition between correct and incorrect loop-loop base-pairs, decrease the rate at which the active pre-RNA structure is assembled.

Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA.

Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.

Folding of RNA involves parallel pathways.

Folding kinetics of large RNAs are just beginning to be investigated. We show that the Tetrahymena self-splicing RNA partitions into a population that rapidly reaches the native state, and a slowly folding population that is trapped in metastable misfolded structures. Transitions from the misfolded structures to the native state involve partial unfolding. The total yield of native RNA is increased by iterative annealing of the inactive population, and mildly denaturing conditions increase the rate of folding at physiological temperatures. These results provide the first evidence that an RNA can fold by multiple parallel paths.

Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics.

Condensed counterions contribute to the stability of compact structures in RNA, largely by reducing electrostatic repulsion among phosphate groups. Varieties of cations induce a collapsed state in the Tetrahymena ribozyme that is readily transformed to the catalytically active structure in the presence of Mg2+. Native gel electrophoresis was used to compare the effects of the valence and size of the counterion on the kinetics of this transition. The rate of folding was found to decrease with the charge of the counterion. Transitions in monovalent ions occur 20- to 40-fold faster than transitions induced by multivalent metal ions. These results suggest that multivalent cations yield stable compact structures, which are slower to reorganize to the native conformation than those induced by monovalent ions. The folding kinetics are 12-fold faster in the presence of spermidine3+ than [Co(NH3)6]3+, consistent with less effective stabilization of long-range RNA interactions by polyamines. Under most conditions, the observed folding rate decreases with increasing counterion concentration. In saturating amounts of counterion, folding is accelerated by addition of urea. These observations indicate that reorganization of compact intermediates involves partial unfolding of the RNA. We find that folding of the ribozyme is most efficient in a mixture of monovalent salt and Mg2+. This is attributed to competition among counterions for binding to the RNA. The counterion dependence of the folding kinetics is discussed in terms of the ability of condensed ions to stabilize compact structures in RNA.

Role of counterion condensation in folding of the Tetrahymena ribozyme. I. Equilibrium stabilization by cations.

Folding of RNA into an ordered, compact structure requires substantial neutralization of the negatively charged backbone by positively charged counterions. Using a native gel electrophoresis assay, we have examined the effects of counterion condensation upon the equilibrium folding of the Tetrahymena ribozyme. Incubation of the ribozyme in the presence of mono-, di- and trivalent ions induces a conformational state that is capable of rapidly forming the native structure upon brief exposure to Mg2+. The cation concentration dependence of this transition is directly correlated with the charge of the counterion used to induce folding. Substrate cleavage assays confirm the rapid onset of catalytic activity under these conditions. These results are discussed in terms of classical counterion condensation theory. A model for folding is proposed which predicts effects of charge, ionic radius and temperature on counterion-induced RNA folding transitions.

Persistence length changes dramatically as RNA folds.

We determine the persistence length l(p) for a bacterial group I ribozyme as a function of concentration of monovalent and divalent cations by fitting the distance distribution functions P(r) obtained from small angle x-ray scattering intensity data to the asymptotic form of the calculated P(WLC)(r) for a wormlike chain. The l(p) values change dramatically over a narrow range of Mg(2+) concentration from approximately 21 Angstroms in the unfolded state (U) to approximately 10 Angstroms in the compact (I(C)) and native states. Variations in l(p) with increasing Na(+) concentration are more gradual. In accord with the predictions of polyelectrolyte theory we find l(p) alpha 1/kappa(2) where kappa is the inverse Debye-screening length.

Exon sequences distant from the splice junction are required for efficient self-splicing of the Tetrahymena IVS.

The presence of a natural rRNA secondary structure element immediately preceding the 5' splice site of the Tetrahymena IVS can inhibit self-splicing by competing with base pairing between the 5' exon and the guide sequence of the IVS (P1). Formation of this alternative hairpin is preferred in short precursor RNAs, and results in loss of G-addition to the 5' splice site. Pre-rRNAs which contain longer exons of ribosomal sequence, however, splice rapidly. As many as 146 nucleotides of the 5' exon and 86 nucleotides of the 3' exon are required for efficient self-splicing of Tetrahymena precursors. The presence of nucleotides distant from the 5' splice site apparently alters the equilibrium between the alternative hairpins, and promotes formation of active precursors. This effect is dependent on the specific sequences of the ribosomal pre-RNA, since point mutations within this region reduce the rate of splicing as much as 50-fold. This system provides an opportunity to study the way in which long-range interactions can influence splice site selection in a highly structured RNA.

Folding mechanisms of group I ribozymes: role of stability and contact order.

The mechanism by which RNA molecules assemble into unique three-dimensional conformations is important for understanding their function, regulation and interactions with substrates. The Tetrahymena group I ribozyme is an excellent model system for understanding RNA folding mechanisms, because the catalytic activity of the native RNA is easily measured. Folding of the Tetrahymena ribozyme is dominated by intermediates in which the stable P4-P6 domain is correctly formed, but the P3-P9 domain is partially misfolded. The propensity of the RNA to misfold depends on the relative stability of native and non-native interactions. Circular permutation of the Tetrahymena ribozyme shows that the distance in the primary sequence between native interactions also influences the folding pathway.