Force-induced misfolding in RNA

RNA folding is a kinetic process governed by the competition of a large number of structures stabilized by the transient formation of base pairs that may induce complex folding pathways and the formation of misfolded structures. Despite of its importance in modern biophysics, the current understanding of RNA folding kinetics is limited by the complex interplay between the weak base-pair interactions that stabilize the native structure and the disordering effect of thermal forces. The possibility of mechanically pulling individual molecules offers a new perspective to understand the folding of nucleic acids. Here we investigate the folding and misfolding mechanism in RNA secondary structures pulled by mechanical forces. We introduce a model based on the identification of the minimal set of structures that reproduce the patterns of force-extension curves obtained in single molecule experiments. The model requires only two fitting parameters: the attempt frequency at the level of individual base pairs and a parameter associated to a free energy correction that accounts for the configurational entropy of an exponentially large number of neglected secondary structures. We apply the model to interpret results recently obtained in pulling experiments in the three-helix junction S15 RNA molecule (RNAS15). We show that RNAS15 undergoes force-induced misfolding where force favors the formation of a stable non-native hairpin. The model reproduces the pattern of unfolding and refolding force-extension curves, the distribution of breakage forces and the misfolding probability obtained in the experiments.Comment: 28 pages, 11 figure

Unifying thermodynamic and kinetic descriptions of single-molecule processes: RNA unfolding under tension

We use mesoscopic non-equilibrium thermodynamics theory to describe RNA unfolding under tension. The theory introduces reaction coordinates, characterizing a continuum of states for each bond in the molecule. The unfolding considered is so slow that one can assume local equilibrium in the space of the reaction coordinates. In the quasi-stationary limit of high sequential barriers, our theory yields the master equation of a recently proposed sequential-step model. Non-linear switching kinetics is found between open and closed states. Our theory unifies the thermodynamic and kinetic descriptions and offers a systematic procedure to characterize the dynamics of the unfolding processComment: 13 pages, 3 figure

A two-state kinetic model for the unfolding of single molecules by mechanical force

We investigate the work dissipated during the irreversible unfolding of single molecules by mechanical force, using the simplest model necessary to represent experimental data. The model consists of two levels (folded and unfolded states) separated by an intermediate barrier. We compute the probability distribution for the dissipated work and give analytical expressions for the average and variance of the distribution. To first order, the amount of dissipated work is directly proportional to the rate of application of force (the loading rate), and to the relaxation time of the molecule. The model yields estimates for parameters that characterize the unfolding kinetics under force in agreement with those obtained in recent experimental results (Liphardt, J., et al. (2002) {\em Science}, {\bf 296} 1832-1835). We obtain a general equation for the minimum number of repeated experiments needed to obtain an equilibrium free energy, to within $k_BT$, from non-equilibrium experiments using the Jarzynski formula. The number of irreversible experiments grows exponentially with the ratio of the average dissipated work, \bar{\Wdis}, to $k_BT$.}Comment: PDF file, 5 page

Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding

Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechano-chemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Whereever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly, the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.Comment: This is an author-created, un-copyedited version of an article accepted for publication in Physical Biology. IOP Publishing Ltd is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. The definitive publisher authenticated version is available online at DOI:10.1088/1478-3975/8/2/02600

Use of analysis and processing of digital images for evaluation and control of animal behavior in hot climates

The world production of meats is mainly concentrated in the countries with a hot climate. Among these countries, Brazil, considered "world's breadbasket", is located in the intertropical zone, with hot climates and presents lower thermal amplitude. Brazilian poultry is a leader in the production and export of broilers. Brazil has the second largest cattle herd in the world, has the largest commercial herd besides being the largest exporter of beef. Brazilian swine breeding, among the most advanced production chains in the world, occupy the position of fourth largest producer of pork in the world. Projections for Brazil's meat production sector indicate strong growth in the coming years, with countries in hot climates that will continue to sustain future growth in world meat production, ensuring food security in many countries. One of the factors responsible for the success or failure of animal production is the environment, defined by the sum of all physical and biological factors that affect animals. In these regions of hot climates, climatic factors are among the main limiting factors to the development of animal production, which may compromise animal welfare and productivity indices. Behavioral information can aid in the analysis of problems arising from environmental conditions unfavorable to animals, helping both decision making and the use of different environmental conditioning systems. The introduction of technification in the animal behavior evaluation processes, through the use of video cameras and image processing programs, allowed a better interpretation of the behavioral responses, quickly, accurately and non-invasively, gradually being used with greater frequency in the animal production sectors. The study of animal behavior assumes an important role in animal production, since, to rationalize the breeding methods, management, feeding and facilities techniques have been developed that interfere with animal behavior. In this context, the use of digital image analysis techniques, where the animals themselves are used as biosensors in response to environmental conditions, contributes to the analysis of animal behavior, and consequently also to the assessment of the internal environment of the production in hot weather. Keywords: animal production, animal welfare, information technolog

Zero Temperature Properties of RNA Secondary Structures

We analyze different microscopic RNA models at zero temperature. We discuss both the most simple model, that suffers a large degeneracy of the ground state, and models in which the degeneracy has been remove, in a more or less severe manner. We calculate low-energy density of states using a coupling perturbing method, where the ground state of a modified Hamiltonian, that repels the original ground state, is determined. We evaluate scaling exponents starting from measurements of overlaps and energy differences. In the case of models without accidental degeneracy of the ground state we are able to clearly establish the existence of a glassy phase with $\theta \simeq 1/3$.Comment: 20 pages including 9 eps figure

Statistical mechanics of RNA folding: importance of alphabet size

We construct a minimalist model of RNA secondary-structure formation and use it to study the mapping from sequence to structure. There are strong, qualitative differences between two-letter and four or six-letter alphabets. With only two kinds of bases, there are many alternate folding configurations, yielding thermodynamically stable ground-states only for a small set of structures of high designability, i.e., total number of associated sequences. In contrast, sequences made from four bases, as found in nature, or six bases have far fewer competing folding configurations, resulting in a much greater average stability of the ground state.Comment: 7 figures; uses revtex

Statistical mechanics of secondary structures formed by random RNA sequences

The formation of secondary structures by a random RNA sequence is studied as a model system for the sequence-structure problem omnipresent in biopolymers. Several toy energy models are introduced to allow detailed analytical and numerical studies. First, a two-replica calculation is performed. By mapping the two-replica problem to the denaturation of a single homogeneous RNA in 6-dimensional embedding space, we show that sequence disorder is perturbatively irrelevant, i.e., an RNA molecule with weak sequence disorder is in a molten phase where many secondary structures with comparable total energy coexist. A numerical study of various models at high temperature reproduces behaviors characteristic of the molten phase. On the other hand, a scaling argument based on the extremal statistics of rare regions can be constructed to show that the low temperature phase is unstable to sequence disorder. We performed a detailed numerical study of the low temperature phase using the droplet theory as a guide, and characterized the statistics of large-scale, low-energy excitations of the secondary structures from the ground state structure. We find the excitation energy to grow very slowly (i.e., logarithmically) with the length scale of the excitation, suggesting the existence of a marginal glass phase. The transition between the low temperature glass phase and the high temperature molten phase is also characterized numerically. It is revealed by a change in the coefficient of the logarithmic excitation energy, from being disorder dominated to entropy dominated.Comment: 24 pages, 16 figure

TRACE ELEMENTS IN SHEEP AND GOATS REPRODUCTION: A REVIEW

The reproduction of small ruminants like goats and sheep managed under extensive range grazing conditions can be affected by nutrients availability and especially by the mineral content of the forages resources on the rangeland. It has been particularly demonstrated that trace elements can have equally, beneficial or detrimental effects, depending on its balance, on reproductive functions in small ruminants. Trace elements as copper, molybdenum, selenium and zinc play key role on the metabolism of carbohydrates, proteins and lipids; however, the mode of action by which these elements affect reproduction in sheep and goats are not completely understood, due to the complexity in the mode of action of the metallobiomolecules and the neuro-hormonal relationship. In this way, their absence or presence of these minerals in several organs, fluids, or tissues of the reproductive tract have allowed obtaining information on the metabolism and the role of these elements on reproduction in sheep and goats. On this regard, the objective of this document is to review the relationships and effects of some trace elements, on reproductive events in sheep and goats