Evidence for the Universal Scaling Behaviour of a Freely Relaxing DNA Molecule

Relaxation measurements on a fluorescently labelled free DNA molecule after stretching by a Poiseuille flow in a capillary vessel reveal universal scaling features: at intermediate times the scaling exponent of the decay law for the molecule length as a function of time is found to be 0.51 +/- 0.05. This law is in agreement with the prediction of the Brochard-Wyart "stem and flower" model for the relaxation of a stretched polymer chain.Molecular and Cellular BiologyPhysic

Probing complex RNA structures by mechanical force

RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpretated by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.Comment: 9 pages, 9 figure

Molecular transport and flow past hard and soft surfaces: Computer simulation of model systems

The properties of polymer liquids on hard and soft substrates are investigated by molecular dynamics simulation of a coarse-grained bead-spring model and dynamic single-chain-in-mean-field (SCMF) simulations of a soft, coarse-grained polymer model. Hard, corrugated substrates are modelled by an FCC Lennard-Jones solid while polymer brushes are investigated as a prototypical example of a soft, deformable surface. From the molecular simulation we extract the coarse-grained parameters that characterise the equilibrium and flow properties of the liquid in contact with the substrate: the surface and interface tensions, and the parameters of the hydrodynamic boundary condition. The so-determined parameters enter a continuum description like the Stokes equation or the lubrication approximation.Comment: 41 pages, 13 figure

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Repression of Mitochondrial Translation, Respiration and a Metabolic Cycle-Regulated Gene, SLF1, by the Yeast Pumilio-Family Protein Puf3p

Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3′-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity

Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries

<p>Abstract</p> <p>Background</p> <p>Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of <it>Streptococcus mutans </it>(implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of <it>S. mutans</it>.</p> <p>Methods</p> <p>A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of <it>S. mutans </it>using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms.</p> <p>Results</p> <p>All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of <it>S. mutans </it>Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of <it>S. mutans </it>strains Ingbritt and NG8 and <it>Lactococcus lactis </it>expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins.</p> <p>Conclusion</p> <p>Gp-340 I behaves as a caries susceptibility protein.</p

Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p

The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay. Using integrated genome-wide approaches we define an expanded set of Puf3p target mRNAs and quantitatively assessed the global impact of loss of PUF3 on gene expression using mRNA and polysome profiling and quantitative proteomics. In agreement with prior studies, our sequencing of affinity-purified Puf3-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. Additionally, we also found 720  new mRNA targets that predominantly encode proteins that enter the nucleus. Comparing transcript levels in wild-type and puf3∆ cells revealed that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only a limited subset of its target mRNAs. Finally, proteomic and translatomic studies suggest that loss of Puf3p has widespread, but modest, impact on mRNA translation. Taken together our integrated multi-omics data point to multiple classes of Puf3p targets, which display coherent post-transcriptional regulatory properties and suggest Puf3p plays a broad, but nuanced, role in the fine-tuning of gene expression

Structural transitions in DNA driven by external force and torque.

Experiments on single DNA molecules have shown that abrupt transitions between states of different extensions can be driven by stretching and twisting. Here we show how a simple statistical-mechanical model can be used to globally fit experimental force-extension data of L?r et al. [Phys. Rev. Lett. 83, 1066 (1999)], over a wide range of DNA molecule twisting. We obtain the mean twists, extensions, and free energies of the five DNA states found experimentally. We also predict global force-torque and force-linking number phase diagrams for DNA. At zero force, the unwinding torque for zero-force structural transition from the double helix to an unwound structure is found to be approximately -2kBT, while the right-handed torque needed to drive DNA to a highly overwound state approximately 7kBT