Thermodynamics of unfolding mechanisms of mouse mammary tumor virus pseudoknot from a coarse-grained loop-entropy model.

Pseudoknotted RNA molecules play important biological roles that depend on their folded structure. To understand the underlying principles that determine their thermodynamics and folding/unfolding mechanisms, we carried out a study on a variant of the mouse mammary tumor virus pseudoknotted RNA (VPK), a widely studied model system for RNA pseudoknots. Our method is based on a coarse-grained discrete-state model and the algorithm of PK3D (pseudoknot structure predictor in three-dimensional space), with RNA loops explicitly constructed and their conformational entropic effects incorporated. Our loop entropy calculations are validated by accurately capturing previously measured melting temperatures of RNA hairpins with varying loop lengths. For each of the hairpins that constitutes the VPK, we identified alternative conformations that are more stable than the hairpin structures at low temperatures and predicted their populations at different temperatures. Our predictions were validated by thermodynamic experiments on these hairpins. We further computed the heat capacity profiles of VPK, which are in excellent agreement with available experimental data. Notably, our model provides detailed information on the unfolding mechanisms of pseudoknotted RNA. Analysis of the distribution of base-pairing probability of VPK reveals a cooperative unfolding mechanism instead of a simple sequential unfolding of first one stem and then the other. Specifically, we find a simultaneous "loosening" of both stems as the temperature is raised, whereby both stems become partially melted and co-exist during the unfolding process.

"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy.

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). A previous Forster resonance energy transfer (FRET) study mapped DNA conformational distributions with cytosine analog FRET pair primarily sensitive to DNA twisting/unwinding deformations (Chakraborty et al. Nucleic Acids Res. 46: 1240-1255 (2018)). These studies revealed B-DNA conformations for nonspecific (matched) constructs but significant unwinding for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with Atto550/Atto647N FRET dyes suitable for fluorescence correlation spectroscopy (FCS). With these probes, we detected higher FRET in specific, mismatched DNA compared with matched DNA, reaffirming unwinding/bending deformations in mismatched DNA. FCS unveiled the dynamics of these spontaneous deformations at ~ 300 µs with no fluctuations detected for matched DNA within the ~ 600 ns-10 ms FCS time window. These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the < 500 µs "stepping" times of repair proteins on DNA. Such "flexible hinge" dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition.

Impact of DNA sequences on DNA 'opening' by the Rad4/XPC nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding ('opening') the damaged DNA site. Such 'opening' is also observed on a normal DNA sequence containing consecutive C/G's (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA 'opening' could occur on any DNA or if certain structures/sequences would resist being 'opened'. Here, we report that DNA containing alternating C/G's (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote 'opening' contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being 'opened' even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion's mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities. Here we report that photocleavable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically recognizes diverse helix-destabilizing/distorting lesions including bulky organic adduct lesions and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding causes it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair.

Tethering-facilitated DNA 'opening' and complementary roles of ß-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively 'opening' these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a 'kinetic gating' mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how 'opening' is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed 'open' undamaged DNA in solution and that such 'opening' can largely occur without one or the other of the ß-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound 'open' DNA adopts multiple conformations in solution notwithstanding the DNA's original structure or the ß-hairpins. Molecular dynamics simulations reveal compensatory roles of the ß-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA 'opening' of undamaged sites and the dynamic nature of 'open' DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

Two new species of braconid wasps (Hymenoptera, Braconidae) from India.

Two new species viz., Pambolus (Phaenodus) shujaisp. nov., and Parachremylus trachysi sp. nov., of braconid wasps are described as new to science. Parachremylus trachysi sp. nov., is reared from larvae of the leaf miner Trachys sp. (Coleoptera, Buprestidae) on Corchorus sp. (Wild Jute Plant). A new species of Pambolus Haliday along with two known species is also recorded. A key to the Indian species of Pambolus is also provided. Diagnoses with morphological characters and illustrations are provided.

Evidence for a bind-then-bend mechanism for architectural DNA binding protein yNhp6A.

The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are 'captured' by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to ∼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to ∼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a-DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 µs-1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.

Static Kinks or Flexible Hinges: Multiple Conformations of Bent DNA Bound to Integration Host Factor Revealed by Fluorescence Lifetime Measurements.

Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations, including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H') into a U-turn by kinking DNA at two sites. We utilized fluorescence-lifetime-based FRET spectroscopy to assess the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 78% of the IHF-H' population exhibited FRET efficiency consistent with the crystal structure, 22% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks the A-tract of H' found on one side of the binding site, the extent of bending in the fully U-bent conformation decreased, and the population in that state decreased to 32%. A similar decrease in the U-bent population was observed with a single base mutation in H' in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not) and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H' and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability, and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary tumor virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly.

Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a µs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 µs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating 'conformational capture' of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a ß-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-µs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The ß-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC.

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor.

Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SK(bi) ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SK(uni) < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

Exploring the energy landscape of nucleic acid hairpins using laser temperature-jump and microfluidic mixing.

We have investigated the multidimensionality of the free energy landscape accessible to a nucleic acid hairpin by measuring the relaxation kinetics in response to two very different perturbations of the folding/unfolding equilibrium, either a laser temperature-jump or ion-jump (from rapid mixing with counterions). The two sets of measurements carried out on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine loop), using FRET between end labels or fluorescence of 2-aminopurine in the stem as conformational probes, yield distinctly different relaxation kinetics in the temperature range 10-30 °C and salt range 100-500 mM NaCl, with rapid mixing exhibiting slower relaxation kinetics after an initial collapse of the chain within 8 µs of the counterion mixing time. The discrepancy in the relaxation times increases with increasing temperatures, with rapid mixing times nearly 10-fold slower than T-jump times at 30 °C. These results rule out a simple two-state scenario with the folded and unfolded ensemble separated by a significant free energy barrier, even at temperatures close to the thermal melting temperature T(m). Instead, our results point to the scenario in which the conformational ensemble accessed after counterion condensation and collapse of the chain is distinctly different from the unfolded ensemble accessed with T-jump perturbation. Our data suggest that, even at temperatures in the vicinity of T(m) or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation.

Mapping the transition state for DNA bending by IHF.

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, "prebent" DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.

A kinetic zipper model with intrachain interactions applied to nucleic acid hairpin folding kinetics.

Single-stranded DNA and RNA hairpin structures with 4-10 nucleotides (nt) in the loop and 5-8 basepairs (bp) in the stem fold on 10-100 µs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ~2-nt) is 10-100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔG(nWC)) and the rate at which a basepair is formed adjacent to an existing basepair (k(bp)(+)). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4-20-nt-long poly(dT) loops, with ΔG(nWC) ≈ -2.4 kcal/mol and k(bp)(+) ≥ (1 ns)(-1), in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.

Fast folding of RNA pseudoknots initiated by laser temperature-jump.

RNA pseudoknots are examples of minimal structural motifs in RNA with tertiary interactions that stabilize the structures of many ribozymes. They also play an essential role in a variety of biological functions that are modulated by their structure, stability, and dynamics. Therefore, understanding the global principles that determine the thermodynamics and folding pathways of RNA pseudoknots is an important problem in biology, both for elucidating the folding mechanisms of larger ribozymes as well as addressing issues of possible kinetic control of the biological functions of pseudoknots. We report on the folding/unfolding kinetics of a hairpin-type pseudoknot obtained with microsecond time-resolution in response to a laser temperature-jump perturbation. The kinetics are monitored using UV absorbance as well as fluorescence of extrinsically attached labels as spectroscopic probes of the transiently populated RNA conformations. We measure folding times of 1-6 ms at 37 °C, which are at least 100-fold faster than previous observations of very slow folding pseudoknots that were trapped in misfolded conformations. The measured relaxation times are remarkably similar to predictions of a computational study by Thirumalai and co-workers (Cho, S. S.; Pincus, D.L.; Thirumalai, D. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17349-17354). Thus, these studies provide the first observation of a fast-folding pseudoknot and present a benchmark against which computational models can be refined.

New insights into the transition pathway from nonspecific to specific complex of DNA with Escherichia coli integration host factor.

To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate favorable interactions and that contribute to the negative enthalpy change, must occur as the system leaves the transition state, downhill to the final complex.

Loop dependence of the stability and dynamics of nucleic acid hairpins.

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as approximately L(8.5 +/- 0.5), in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl2, the stabilities of both ssDNA and RNA hairpins scale as approximately L(4 +/- 0.5), indicating that the intraloop interactions are weaker in the presence of Mg2+. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl2) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, approximately L(2.2 +/- 0.5) and approximately L(2.6 +/- 0.5), respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na+ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation.