Reza Behrouzi

Structural rearrangements linked to global folding pathways of the Azoarcus group I ribozyme.

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (tau < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg2+, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s-1 at 37 degrees C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the IC intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.

Crowders perturb the entropy of RNA energy landscapes to favor folding.

Biological macromolecules have evolved to fold and operate in the crowded environment of the cell. We have shown previously that molecular crowding stabilizes folded RNA structures. Here we report SAXS measurements on a 64 kDa bacterial group I ribozyme in the presence of mono- and divalent ions and PEG crowders of different molecular weight. These experiments show that crowders always stabilize the folded RNA, but this stabilization is weaker in NaCl solutions than MgCl2 solutions. Additionally, we find that RNAs with the same global structure, parametrized by Rg, have different scattering functions depending upon the ratio of electrostatic and entropic stabilization by ions and crowders, respectively. We quantify this difference using the scattering length per scattering volume and find that this ratio is larger for RNAs that fold in lower ionic strength solutions due to the higher crowder content. We conclude that lower RNA flexibility, or reduced configurational entropy, widens the free energy gap between the unfolded and folded RNA in crowded MgCl2 solutions.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.

Entropic stabilization of folded RNA in crowded solutions measured by SAXS

Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cells interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg2+ concentrations typical of the cells interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl2 and polyethylene glycol (PEG) concentrations. The resulting phase diagrams show that perturbations to folding by each variable do not overlap. A favorable enthalpy change drives the formation of compact, native-like structures, but requires Mg2+ ions at all temperatures studied (5–55°C). PEG reduces the entropic cost of helix assembly and increases correlations between RNA segments at all temperatures. The phase diagrams also revealed a semi-compact intermediate between the unfolded and folded ensemble that is locally more flexible than the unfolded state, as judged by SHAPE modification. These results suggest that environmental variables such as temperature and solute density will favor different types of RNA structures.

Effects of Preferential Counterion Interactions on the Specificity of RNA Folding

The real-time search for native RNA structure is essential for the operation of regulatory RNAs. We previously reported that a fraction of the Azoarcus ribozyme achieves a compact structure in less than a millisecond. To scrutinize the forces that drive initial folding steps, we used time-resolved SAXS to compare the folding dynamics of this ribozyme in thermodynamically isostable concentrations of different counterions. The results show that the size of the fast-folding population increases with the number of available counterions and correlates with the flexibility of initial RNA structures. Within 1 ms of folding, Mg2+ exhibits a smaller preferential interaction coefficient per charge, ΔΓ+/ Z, than Na+ or [Co(NH3)6]3+. The lower ΔΓ+/ Z corresponds to a smaller yield of folded RNA, although Mg2+ stabilizes native RNA more efficiently than other ions at equilibrium. These results suggest that strong Mg2+-RNA interactions impede the search for globally native structure during early folding stages.

Rendering RNA in 3D

Integrating biochemical footprinting data with molecular dynamics yields more accurate RNA three-dimensional structure predictions.

The Role of Electrostatic Relaxation on the Folding Kinetics of a Bacterial Ribozyme

The self-assembly of catalytic RNA ribozymes into compact, native structures is critical for their functions in the cell. The first step in forming RNA tertiary structure is the neutralization by cations of the negative charges of the phosphates. This electrostatic stabilization enables dynamical exploration of more compact conformations, and the search for long-range tertiary interactions. Our previous time-resolved Small Angle X-ray Scattering (SAXS) studies showed that the Azoarcus ribozyme exhibits triphasic folding kinetics: up to 90% folds in less than 10 ms, which we attribute to specific collapse.