Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers.

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.

Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB.

Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a 'Helicase and RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, 'slow' and 'normal', as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third 'fast' unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.

Single molecule studies of helicases with magnetic tweezers.

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.

Bioenergy feedstock production in Chlamydomonas reinhardtii (microalgae) cultivated under mixotrophic growth with cellulose hydrolysate from agricultural waste.

In the present study, cellulose purified from finger millet agricultural waste is subjected to enzymatic hydrolysis, and the hydrolysate (predominantly glucose) is used as a carbon source supplement in the media for the mixotrophic growth of Chlamydomonas reinhardtii. Interestingly, a switch between excess starch production and excess lipid (triacylglycerols, TAG) production occurs by a small change in hydrolysate concentration in the media. Starch production increased 4.5-fold with respect to the photoautotrophic control, with a glucose concentration of 3 mg/mL in the media after hydrolysate addition. This culture had TAG production enhancement by 1.5-fold. However, mixotrophic cultivation with 4 mg/mL glucose concentration in the media with hydrolysate addition resulted in TAG productivity enhancement by 4.2-fold compared to control and starch amount increase of 1.3-fold. The organic carbon source (glucose) and the inorganic carbon source (citrate ions) in the hydrolysate together played a role in this delicate switching between starch and lipid pathways. Proteins, starch, and TAG molecules are analyzed in the microalgal cells grown under different conditions with FTIR spectroscopy, a rapid, high-throughput method of biomolecular estimation. High-resolution single-cell AFM studies of the cell wall structure reveal enhanced corrugations in surface morphology during mixotrophic growth with cellulose hydrolysate, illustrating an adaptive mechanism with improved mechanical stress management. Lipid droplet morphology at the single-cell level points to two distinct mechanisms of lipid accumulation: one in which the lipids are segregated as droplets, and the other in which lipid molecules are uniformly dispersed in the cytosol as unresolved, ultra-small droplets. The present study therefore analyzes both the bulk and the single-cell level changes when cellulose hydrolysate is used as a carbon source for Chlamydomonas reinhardtii mixotrophic cultivation, which serves a four-fold purpose: value from waste, fixation of atmospheric CO2, production of lipids for biodiesel, and starch for bioethanol.

Probing Microscale Structuring-Induced Phase Separation with Fluorescence Recovery Diffusion Dynamics in Poly(ethylene glycol) Solutions.

Apart from biocompatibility, poly(ethylene glycol) (PEG)-based biomedical constructs require mechanical tunability and optimization of microscale transport for regulation of the release kinetics of biomolecules. This study illustrates the role of inhomogeneities due to aggregates and structuring in the PEG matrix in the microscale diffusion of a fluorescent probe. Comparative analysis of fluorescence recovery after photobleaching (FRAP) profiles with the help of diffusion half-time is used to assess the diffusion coefficient (D). The observations support a nontrivial dependence of diffusion dynamics on polymer concentration (volume fraction, φ) and that of fillers carboxymethyl cellulose (CMC) and nanoclay bentonite (B). D values follow the Rouse scaling D ∼ φ-0.54 in PEG solutions. The diffusion time of the fluorescent probe in the PEG+bentonite matrix reveals the onset of depletion interaction-induced phase separation with an increase in bentonite concentration in the PEG matrix beyond 0.1 wt %. Beyond this concentration, structure factors obtained from prebleach FRAP images show a rapid increase at low Q. The two-phase system (PEG-rich and bentonite-rich) was characterized by the hierarchical structural topology of bentonite aggregates, and aggregate sizes were obtained at different length scales with phase contrast imaging, small-angle neutron scattering, and small-angle X-ray scattering. The microscale transport detection presented captures sensitively the commencement of phase separation in the PEG + bentonite matrix, as opposed to the PEG or PEG + CMC matrix, which are observed to be one-phase systems. This method of diffusion half-time and prebleach image analysis can be used for the fast, high-throughput experimental investigation of microscale mechanical response and its correlation with structuring in the polymer matrix.

Micro-Raman spectroscopy of the light-harvesting pigments in Chlamydomonas reinhardtii under salinity stress.

Microalgae are a rich source of carotenoids with enhanced yields during biotic or abiotic stresses, which often impose survival challenges on the cells. Using a non-invasive pigment profiling approach with micro-Raman spectroscopy, we have analyzed the effect of salinity stress on carotenoids in autotrophic Chlamydomonas reinhardtii. Raman spectral analysis of ν(C = C) mode indicates an increase in the carotenoids with lower conjugation length (lutein and zeaxanthin) compared to ß-carotene, as the function of culture age and salinity stress, but especially when salinity stress was imposed in two-stage mode (stress imposed on 2nd day, D2_100, and 4th day, D4_100, during exponential phase). Population-scale heterogeneities in carotenoid Raman mode peak center, quantified with heterogeneity index (HI), were highest during the stationary phase of the cultures and under salinity stress. Although the Raman signal was obtained from a randomly selected small focal volume in the cell, a decrease in chlorophyll Raman mode intensities with age and salinity stress was well corroborated by single-cell population fraction measurements by microscopy. Raman intensity fluctuations (If) were high for both chlorophyll and carotenoid modes under salinity stress, which can arise due to variations in chlorophyll/carotenoid content and composition, or conformational changes in the pigments in C. reinhardtii cells. Interestingly, in all growth conditions, chlorophyll a Raman mode intensity was found to show a high correlation to that of ß-carotene, pointing out a high degree of cooperativity in the light-harvesting complex pigments even during salinity stress. Thus, we demonstrate the usefulness of non-invasive pigment profiling with micro-Raman spectroscopy for developing an optimization for salinity stress conditions for high biomass yield and proper harvest time to obtain carotenoids with desired chemical composition.

Molecular and micro-scale heterogeneities in Raman modes of a relaxing polymer glass.

We have used Raman spectroscopy to study relaxation dynamics at two different length scales, molecular level and micro-scale in order to probe the presence of cooperative rearranging regions in a polymer glass. Response to slow thermal cycles and fast quench through the glass transition temperature (Tg) is analyzed for film and unprocessed forms of polyvinyl acetate (PVAc). In PVAc film, enhanced disorder and molecular mobility lead to peak broadening by about a factor of 10 compared to unprocessed PVAc. Thermal cycles (10 K min-1) produce hysteresis in integrated Raman peak intensity (loop areaAINTI).AINTIvalues of film are two orders of magnitude more than unprocessed, indicating more configurational mosaics with higher interfacial energy dissipations. Ageing after 60 K min-1quench manifests as heterogeneous molecular dynamics of film Raman modes with significant peak-width variations, differentiating high mobility and low mobility modes. Two-dimensional mapping of film Raman modes after quench reveal micro-scale clusters of average size ≈250 molecules having fractal boundaries with fractal dimensiondf= 1.5, resemblingdfof percolation clusters below percolation threshold. During thermal cycling and relaxation after a quench, cooperative segmental dynamics with large correlations between skeletal C-C stretch and side branch modes is observed. The observations are analyzed in the context of the random first order transition theory of glasses, which attributes heterogeneous relaxations in glasses to the presence of clusters of variable configurational states.

Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase.

The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.

π-conjugation and conformation in a semiconducting polymer: small angle x-ray scattering study.

Small angle x-ray scattering (SAXS) in a poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) solution has shown the important role of π-electron conjugation in controlling the chain conformation and assembly. By increasing the extent of conjugation from 30 to 100%, the persistence length (l(p)) increases from 20 to 66 Å. Moreover, a pronounced second peak in the pair distribution function has been observed in a fully conjugated chain, at larger length scales. This feature indicates that the chain segments tend to self-assemble as the conjugation along the chain increases. Xylene enhances the rigidity of the PPV backbone to yield extended structures, while tetrahydrofuran solvates the side groups to form compact coils in which the l(p) is much shorter.

A mechanistic study of helicases with magnetic traps.

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.

Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities.

RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery.

Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V(un)) by the velocity of translocation on ssDNA in nucleotides per second (V(trans)). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the "active" range. In the case of Dda, the average DNA unwinding velocity was 257±42 bp/s, and the average translocation velocity was 267±15 nt/s. The V(un)/V(trans) value of 0.96 places Dda in a unique category of being an essentially "perfectly" active helicase.

Ion-induced multiply reentrant liquid-liquid transitions and the nature of criticality in ethanol-water mixture.

We report a quite unusual feature of four liquid-liquid reentrant transitions in ethanol (E)+water (W)+ammonium sulfate mixture by meticulous tuning of the ammonium sulfate concentration in a narrow range, as a function of temperature, at atmospheric pressure. Detailed exploration of the intricate phase behavior in terms of E/W sections shows that the range of triple reentrance shrinks with increasing E/W. The behavior of osmotic susceptibility is investigated by light scattering, near the critical point, in the one-phase region by varying the temperature at fixed concentration of the components, in a particular E/W section. The critical exponent of susceptibility (gamma) and correlation length (nu) are observed to have Fisher renormalized Ising values [Phys. Rev. 176, 237 (1968)], with gamma(r)=1.41 and nu(r)=0.718. The effective susceptibility exponent, gamma(eff), exhibits a sharp, nonmonotonic crossover from Ising to mean-field critical behavior, which is completed outside the critical regime. The amplitude of the correlation length, xi(o)(=21.2+/-0.4 A), deduced from light scattering experiment, is an order of magnitude larger than the typical values in usual aqueous electrolyte systems. This value of xi(o) is further verified from small-angle x-ray scattering (SAXS) experiments and found to be consistent. SAXS experiments on the critical sample reveal the presence of long-ranged intermolecular correlations, leading to supramolecular structuring, at a temperature far away from the critical point. These results convincingly demonstrate that the finite length scale arising due to the structuring competes with the diverging correlation length of critical concentration fluctuations, which influences the nonasymptotic critical behavior in this aqueous electrolyte system. The sulphate ions play a dominant role in both structuring and the complex phase behavior.