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 experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, 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, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The 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. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.Comment: Supporting Information include

Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition

Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition Many cellular processes involve interactions between proteins and DNA in which proteins recognize and bind to specific sites on the DNA with thousand- or million-fold higher affinities than to random DNA sequences. How these proteins search for and find their specific sites in genomic DNA amidst a large excess (~3 billion) of nonspecific sites remains a puzzle. Many site-specific proteins kink, bend or twist DNA at that site, and undergo concerted conformational rearrangements to accommodate the deformed DNA (‘induced-fit mechanism’). In many cases, the proteins discriminate between specific and nonspecific sites primarily by sensing differences in local DNA deformability (‘indirect readout’), rather than by relying on direct interactions with target nucleotides. How rapidly the deformations occur during target recognition and how they compare with the time that a searching protein spends on a given DNA site before diffusing away remain largely unknown, obscuring our understanding of target recognition mechanisms. Site-specific recognition is expected to be fast or comparable to the protein’s ‘residence time’ per DNA site. Direct observations of proteins undergoing one-dimensional diffusion on nonspecific DNA indicate stepping times (or residence times) per base pair ranging from 50 ns 500 s, considerably shorter than the ~10 ms timescales previously reported for DNA conformational dynamics during binding-site recognition. This posed a puzzle, and suggested that previous studies were likely not resolving key dynamical steps that led to target recognition. I will present recent results on DNA conformational dynamics for three specific protein-DNA complexes: (1) IHF: a prokaryotic architectural protein that recognizes and severely bends specific sites on -phage DNA into a U-turn; (2) XPC: a DNA repair protein that recognizes bulky lesions, unwinds the DNA at that site, and flips out the damaged nucleotides; (3) MutS: another DNA repair protein that recognize mismatches in DNA and sharply kinks the DNA at the mismatched site. All three proteins rely primarily on indirect readout to recognize their DNA target site and therefore must have the ability to sense and discern sequence-dependent DNA deformability while rapidly scanning DNA in search for their target sites. Using nanosecond laser temperature-jump perturbation approach in combination with novel fluorescent probes that enabled protein-DNA dynamics to be measured on timescales of 20 s to > 50 ms, my studies have uncovered previously unresolved steps in the recognition process, including rapid (sub-ms) DNA bending and unwinding that are commensurate with rapid searching while nonspecifically bound, and slower specific recognition steps such as nucleotide flipping or severe DNA bending to form a tight fit. These kinetics measurements help illuminate how a searching protein interrogates DNA deformability and eventually ‘stumbles’ upon its target site, revealing rich multi-step dynamics during this search-interrogation-recognition process

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 Tm. 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 Tm or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation

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