scispace - formally typeset
Search or ask a question

Showing papers by "Carlos Bustamante published in 2003"


Journal ArticleDOI
23 Jan 2003-Nature
TL;DR: The basic features of DNA were elucidated during the half-century following the discovery of the double helix, but it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties.
Abstract: The basic features of DNA were elucidated during the half-century following the discovery of the double helix. But it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties. These studies have illuminated the nature of interactions between DNA and proteins, the constraints within which the cellular machinery operates, and the forces created by DNA-dependent motors.

1,254 citations


Journal ArticleDOI
17 Jul 2003-Nature
TL;DR: Tests of the linearity of DNA's twist elasticity, direct measurements of the torsional modulus, characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes are reported.
Abstract: Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA 1‐3 have allowed direct determination of the molecule’s mechanical properties, provided rigorous tests of theories of polymer elasticity 4 , revealed unforeseen structural transitions induced by mechanical stresses 3,5‐7 , and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA 8 . However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments. We have measured torque as a function of twist for stretched DNA—torsional strain in over- or underwound molecules was used to power the rotation of submicrometre beads serving as calibrated loads. Here we report tests of the linearity of DNA’s twist elasticity, direct measurements of the torsional modulus (finding a value ,40% higher than generally accepted), characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes. We also show that cooperative structural transitions in DNA can be exploited to construct constanttorque wind-up motors and force‐torque converters. Previous investigations of the force‐extension behaviour of supercoiled DNA have found that, under low tension, DNA behaves like an isotropic flexible rod 2,9 : as turns are added to the molecule, its

545 citations


Book ChapterDOI
TL;DR: The optical-trap force transducer that operates by the direct measurement of light momentum is described, which is useful in molecular and cell biology because several important forces are in an accessible piconewton (pN) range.
Abstract: Publisher Summary This chapter describes the optical-trap force transducer that operates by the direct measurement of light momentum. By focusing a laser beam through a microscope objective, it has been found that particles with high indexes of refraction—such as glass, plastic, or oil droplets—are attracted to intense regions in the beam and could be held permanently at a focal point. Optical tweezers are useful in molecular and cell biology because several important forces are in an accessible piconewton (pN) range—for example, ligand-receptor binding, DNA stretching, protein unfolding, and molecular motor stall forces. Methods to measure such forces in the optical trap are under continuous development. The position of a spherical bead inside a trap can be measured with subnanometer precision by one of several optical methods, and so this technique has proven very effective. Methods to measure such forces in the optical trap have been under continuous development. Most force-measurement methods treat the optical trap as a harmonic potential well or virtual spring that pulls the bead toward the trap center.

327 citations


Journal ArticleDOI
TL;DR: The large N limit is derived for the Jarzynski estimator bias, variance, and mean square error that is correct for arbitrary perturbations and shows that, for near-equilibrium switching, the Jarzyk estimator is always superior to the mean work estimator and is even superior toThe fluctuation–dissipation estimator for small N.
Abstract: In 1997, Jarzynski proved a remarkable equality that allows one to compute the equilibrium free-energy difference DeltaF between two states from the probability distribution of the nonequilibrium work W done on the system to switch between the states, e-DeltaF/kappaT =e-W/kappaT [Jarzynski, C. (1997) Phys. Rev. Lett. 87, 2690-2693]. The Jarzynski equality provides a powerful free-energy difference estimator from a set of N irreversible experiments and is closely related to free-energy perturbation, a common computational technique for estimating free-energy differences. Despite the many applications of the Jarzynski estimator, its behavior is only poorly understood. In this article we derive the large N limit for the Jarzynski estimator bias, variance, and mean square error that is correct for arbitrary perturbations. We then analyze the properties of the Jarzynski estimator for all N when the probability distribution of work values is Gaussian, as occurs, for example, in the near-equilibrium regime. This allows us to quantitatively compare it to two other free-energy difference estimators: the mean work estimator and the fluctuation-dissipation theorem estimator. We show that, for near-equilibrium switching, the Jarzynski estimator is always superior to the mean work estimator and is even superior to the fluctuation-dissipation estimator for small N. The Jarzynski-estimator bias is shown to be the dominant source of error in many cases. Our expression for the bias is used to develop a bias-corrected Jarzynski free-energy difference estimator in the near-equilibrium regime.

314 citations


Journal ArticleDOI
21 Mar 2003-Science
TL;DR: The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.
Abstract: Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are "brittle," breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

244 citations


Journal ArticleDOI
TL;DR: It is shown that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe, which indicates a preference for a single-crossing geometry during strand passage.
Abstract: Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils. Here, we investigate the mechanism underlying this chiral preference. Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions. Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle.

173 citations


Journal ArticleDOI
TL;DR: In both humans and chimpanzees, OR pseudogenes seem to evolve neutrally, whereas, in humans, there is suggestive evidence for positive selection acting on intact OR genes, which is likely due to differences in lifestyle, between humans and great apes, that have led to distinct sensory needs.
Abstract: The olfactory receptor (OR) genes constitute the largest gene family in mammalian genomes. Humans have >1,000 OR genes, of which only ∼40% have an intact coding region and are therefore putatively functional. In contrast, the fraction of intact OR genes in the genomes of the great apes is significantly greater (68%–72%), suggesting that selective pressures on the OR repertoire vary among these species. We have examined the evolutionary forces that shaped the OR gene family in humans and chimpanzees by resequencing 20 OR genes in 16 humans, 16 chimpanzees, and one orangutan. We compared the variation at the OR genes with that at intergenic regions. In both humans and chimpanzees, OR pseudogenes seem to evolve neutrally. In chimpanzees, patterns of variability are consistent with purifying selection acting on intact OR genes, whereas, in humans, there is suggestive evidence for positive selection acting on intact OR genes. These observations are likely due to differences in lifestyle, between humans and great apes, that have led to distinct sensory needs.

152 citations


Journal ArticleDOI
TL;DR: It is found that there is no evidence that a significant fraction of fixed amino acid replacements is neutral or nearly neutral or that positive selection drives amino acid replacement sites at only a subset of the loci.
Abstract: One of the principal goals of population genetics is to understand the processes by which genetic variation within species (polymorphism) becomes converted into genetic differences between species (divergence). In this transformation, selective neutrality, near neutrality, and positive selection may each play a role, differing from one gene to the next. Synonymous nucleotide sites are often used as a uniform standard of comparison across genes on the grounds that synonymous sites are subject to relatively weak selective constraints and so may, to a first approximation, be regarded as neutral. Synonymous sites are also interdigitated with nonsynonymous sites and so are affected equally by genomic context and demographic factors. Hence a comparison of levels of polymorphism and divergence between synonymous sites and amino acid replacement sites in a gene is potentially informative about the magnitude of selective forces associated with amino acid replacements. We have analyzed 56 genes in which polymorphism data from D. simulans are compared with divergence from a reference strain of D. melanogaster. The framework of the analysis is Bayesian and assumes that the distribution of selective effects (Malthusian fitnesses) is Gaussian with a mean that differs for each gene. In such a model, the average scaled selection intensity (γ =N e s) of amino acid replacements eligible to become polymorphic or fixed is −7.31, and the standard deviation of selective effects within each locus is 6.79 (assuming homoscedasticity across loci). For newly arising mutations of this type that occur in autosomal or X-linked genes, the average proportion of beneficial mutations is 19.7%. Among the amino acid polymorphisms in the sample, the expected average proportion of beneficial mutations is 47.7%, and among amino acid replacements that become fixed the average proportion of beneficial mutations is 94.3%. The average scaled selection intensity of fixed mutations is +5.1. The presence of positive selection is pervasive with the single exception of kl-5, a Y-linked fertility gene. We find no evidence that a significant fraction of fixed amino acid replacements is neutral or nearly neutral or that positive selection drives amino acid replacements at only a subset of the loci. These results are model dependent and we discuss possible modifications of the model that might allow more neutral and nearly neutral amino acid replacements to be fixed.

147 citations


Journal ArticleDOI
TL;DR: It is shown that in the limit of a single, short molecule the outcome of experimental measurements may depend on which variables are held fixed and which are allowed to fluctuate, and the basic thermodynamic concepts of an intensive or extensive quantity are less useful for interpreting single-molecule experiments.

98 citations


Journal ArticleDOI
01 Feb 2003-Genetics
TL;DR: Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes.
Abstract: Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (omega = K(a)/K(s)). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of omega among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (approximately 5%) have an estimated omega > 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci.

74 citations


Journal ArticleDOI
TL;DR: A mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice is discussed in which variations in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the Sarcomeric passive force response.

Journal ArticleDOI
TL;DR: In this article, E.coli RNAP elongation complexes reveal an average DNA compaction of 22nm and a DNA deformation compatible with approximately 180 degrees DNA wrapping against the enzyme, suggesting a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.

Journal ArticleDOI
TL;DR: The framework presented here provides an ideal setting for modeling mutations dispersed through the genome and, in particular, for the analysis of how natural selection operates on different classes of single nucleotide polymorphisms (SNPs).

Journal ArticleDOI
TL;DR: Recent ensemble and single molecule manipulation studies highlight the mechanical nature of protein unfolding in vivo and the greater need to understand the response of proteins to mechanical force.
Abstract: Recent ensemble and single molecule manipulation studies highlight the mechanical nature of protein unfolding in vivo and, thus, the greater need to understand the response of proteins to mechanical force.