Charles Limouse

The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3′3′ ends differs from that which results when it is pulled from the 5′5′ ends. In this work, we demonstrate, using λ phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3′3′ pulling versus 5′5′ pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 ± 3 pN and 122 ± 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 ± 3 pN for 3′3′ and 110 ± 3 pN for 5′5′; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 ± 0.8 pN μm for 3′3′ versus 13 ± 8 pN for 5′5′; and (iv) 3′3′ and 5′5′ molecules show different changes in hysteresis due to interactions with β-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

Protein flexibility is required for vesicle tethering at the Golgi

The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering. DOI: http://dx.doi.org/10.7554/eLife.12790.001

Dentate gyrus mossy cells control spontaneous convulsive seizures and spatial memory

A way to prevent generalized seizures? Temporal lobe epilepsy is the most common form of epilepsy in adults. Patients have spontaneous seizures and risk developing serious cognitive impairment. Bui et al. studied an animal model of temporal lobe epilepsy (see the Perspective by Scharfman). Selective optogenetic inhibition of dentate gyrus mossy cells increased the likelihood of electrographic seizures generalizing to full behavioral convulsive seizures. Activation of mossy cells reduced the likelihood. Thus, the activity of mossy cells might serve to inhibit seizure propagation. Science, this issue p. 787; see also p. 740 There is a direct relationship between mossy cell activity in the dentate gyrus, convulsive seizures, and spatial memory formation in mice. Temporal lobe epilepsy (TLE) is characterized by debilitating, recurring seizures and an increased risk for cognitive deficits. Mossy cells (MCs) are key neurons in the hippocampal excitatory circuit, and the partial loss of MCs is a major hallmark of TLE. We investigated how MCs contribute to spontaneous ictal activity and to spatial contextual memory in a mouse model of TLE with hippocampal sclerosis, using a combination of optogenetic, electrophysiological, and behavioral approaches. In chronically epileptic mice, real-time optogenetic modulation of MCs during spontaneous hippocampal seizures controlled the progression of activity from an electrographic to convulsive seizure. Decreased MC activity is sufficient to impede encoding of spatial context, recapitulating observed cognitive deficits in chronically epileptic mice.

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway.

Significance Many biological processes, including splicing, translation, and genome maintenance, require structured RNAs to fold into complex three-dimensional shapes. Our current understanding of these processes is based on distilling principles from descriptive folding studies. Moving toward predictive models will require coupling observed structural changes with kinetic and thermodynamic measurements. We have dissected P4-P6 RNA folding through distinct structural states and measured the rate and equilibrium constants for transitions between these states. Common kinetics found for RNA tertiary elements embedded in different structural contexts may help develop predictive folding models. Also, our results suggest that RNA folding may be well described by a model analogous to the diffusion-collision model for protein folding. The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Significance We propose and test predictions of a thermodynamic and kinetic model for RNA tertiary folding that is based on separable energetic contributions of RNA elements. We define these contributions based on the principle features of RNA, and we test the basic predictions of separability by determining whether the energetic contributions of one component are affected by changes in another component. Our results support energetic separability of RNA elements and suggest that it may be possible to deconstruct RNAs into smaller parts that can be studied in isolation such that the individual folding behaviors of these parts can be used to “reconstitute” the folding of the original RNA. Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity—that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔGalign, the probability of aligning tertiary contact partners, and ΔGtert, the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔGHJH) or from changes in the electrostatic environment (ΔG+/−) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔGtert). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔGtert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.

Intramolecular dynamics of single molecules in free diffusion

Biomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such “mesocale” molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway

Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.

Exploring Chromatin Biochemistry with Single-Molecule Fluorescence Diffusometry

We utilize real-time feedback to track individual dye-labeled chromatin fibers undergoing Brownian motion in aqueous buffer, enabling simultaneous recording of fluorescence and hydrodynamic data, providing new insight into conformational dynamics and self-association of nucleosome arrays.

Tracking Fluorescence Correlation Spectroscopy of Individual Biomolecules

I will describe our group’s ongoing research utilizing feedback microscopy to obtain tens-of-seconds per-molecule observation times in solution FCS and FRET assays. Results of recent studies on DNA mechanics will be presented.