A data-driven estimation of the ribosome drop-off rate in S. cerevisiae reveals a correlation with the genes length.

Ribosomes are the molecular machinery that catalyse all the fundamental steps involved in the translation of mRNAs into proteins. Given the complexity of this process, the efficiency of protein synthesis depends on a large number of factors among which ribosome drop-off (i.e. the premature detachment of the ribosome from the mRNA template) plays an important role. However, an in vitro quantification of the extent to which ribosome drop-off occurs is not trivial due to difficulties in obtaining the needed experimental evidence. In this work we focus on the study of ribosome drop-off in Saccharomyces cerevisiae by using 'Ribofilio', a novel software tool that relies on a high sensitive strategy to estimate the ribosome drop-off rate from ribosome profiling data. Our results show that ribosome drop-off events occur at a significant rate also when S. cerevisiae is cultured in standard conditions. In this context, we also identified a correlation between the ribosome drop-off rate and the genes length: the longer the gene, the lower the drop-off rate.

Chain Sliding versus ß-Sheet Formation upon Shearing Single α-Helical Coiled Coils.

Coiled coils (CCs) are key building blocks of biogenic materials and determine their mechanical response to large deformations. Of particular interest is the observation that CC-based materials display a force-induced transition from α-helices to mechanically stronger ß-sheets (αßT). Steered molecular dynamics simulations predict that this αßT requires a minimum, pulling speed-dependent CC length. Here, de novo designed CCs with a length between four to seven heptads are utilized to probe if the transition found in natural CCs can be mimicked with synthetic sequences. Using single-molecule force spectroscopy and molecular dynamics simulations, these CCs are mechanically loaded in shear geometry and their rupture forces and structural responses to the applied load are determined. Simulations at the highest pulling speed (0.01 nm ns-1 ) show the appearance of ß-sheet structures for the five- and six-heptad CCs and a concomitant increase in mechanical strength. The αßT is less probable at a lower pulling speed of 0.001 nm ns-1 and is not observed in force spectroscopy experiments. For CCs loaded in shear geometry, the formation of ß-sheets competes with interchain sliding. ß-sheet formation is only possible in higher-order CC assemblies or in tensile-loading geometries where chain sliding and dissociation are prohibited.

A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity.

Malaria is a global health concern, and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85-Å resolution revealed that 5D5 binds an α-helical epitope in N-CSP with high affinity through extensive shape and charge complementarity and the unusual utilization of an antibody N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites and lack of sporozoite inhibition in vitro and in vivo. Overall, our data do not support the inclusion of the 5D5 N-CSP epitope into the next generation of CSP-based vaccines.

Critical Steps of Plasmodium falciparum Ookinete Maturation.

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro. Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites.

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

Goodness of fit testing in dynamic single-molecule force spectroscopy.

Dynamic single-molecule force spectroscopy (SMFS) is a powerful method to characterize the mechanical stability of biomolecules. We address the problem that the standard manner of reporting the extracted energy landscape parameters does not reveal the intrinsic statistical errors associated with them. This problem becomes particularly relevant when SMFS is used to compare two or more different molecular systems. Here, we propose two methods that allow for a straightforward test of statistical significance. We illustrate the power of the methods by applying them to the experimental results obtained for three dimeric coiled coils of different lengths. Both methods are general and may be applied to any problem involving the fit of models with two correlated parameters.

The Role of SufS Is Restricted to Fe-S Cluster Biosynthesis in Escherichia coli.

In Escherichia coli, two different systems that are important for the coordinate formation of Fe-S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe-S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S-containing proteins, including the MoaA protein, which is involved in the conversion of 5'GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.

A conditional likelihood is required to estimate the selection coefficient in ancient DNA.

Time-series of allele frequencies are a useful and unique set of data to determine the strength of natural selection on the background of genetic drift. Technically, the selection coefficient is estimated by means of a likelihood function built under the hypothesis that the available trajectory spans a sufficiently large portion of the fitness landscape. Especially for ancient DNA, however, often only one single such trajectories is available and the coverage of the fitness landscape is very limited. In fact, one single trajectory is more representative of a process conditioned both in the initial and in the final condition than of a process free to visit the available fitness landscape. Based on two models of population genetics, here we show how to build a likelihood function for the selection coefficient that takes the statistical peculiarity of single trajectories into account. We show that this conditional likelihood delivers a precise estimate of the selection coefficient also when allele frequencies are close to fixation whereas the unconditioned likelihood fails. Finally, we discuss the fact that the traditional, unconditioned likelihood always delivers an answer, which is often unfalsifiable and appears reasonable also when it is not correct.

Predict or classify: The deceptive role of time-locking in brain signal classification.

Several experimental studies claim to be able to predict the outcome of simple decisions from brain signals measured before subjects are aware of their decision. Often, these studies use multivariate pattern recognition methods with the underlying assumption that the ability to classify the brain signal is equivalent to predict the decision itself. Here we show instead that it is possible to correctly classify a signal even if it does not contain any predictive information about the decision. We first define a simple stochastic model that mimics the random decision process between two equivalent alternatives, and generate a large number of independent trials that contain no choice-predictive information. The trials are first time-locked to the time point of the final event and then classified using standard machine-learning techniques. The resulting classification accuracy is above chance level long before the time point of time-locking. We then analyze the same trials using information theory. We demonstrate that the high classification accuracy is a consequence of time-locking and that its time behavior is simply related to the large relaxation time of the process. We conclude that when time-locking is a crucial step in the analysis of neural activity patterns, both the emergence and the timing of the classification accuracy are affected by structural properties of the network that generates the signal.

Degradation Parameters from Pulse-Chase Experiments.

Pulse-chase experiments are often used to study the degradation of macromolecules such as proteins or mRNA. Considerations for the choice of pulse length include the toxicity of the pulse to the cell and maximization of labeling. In the general case of non-exponential decay, varying the length of the pulse results in decay patterns that look different. Analysis of these patterns without consideration to pulse length would yield incorrect degradation parameters. Here we propose a method that constructively includes pulse length in the analysis of decay patterns and extracts the parameters of the underlying degradation process. We also show how to extract decay parameters reliably from measurements taken during the pulse phase.

Quantitative assessment of ribosome drop-off in E. coli.

Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength inE. coli Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases.

Bacteria differently regulate mRNA abundance to specifically respond to various stresses.

Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up- and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs.

Circular analysis in complex stochastic systems.

Ruling out observations can lead to wrong models. This danger occurs unwillingly when one selects observations, experiments, simulations or time-series based on their outcome. In stochastic processes, conditioning on the future outcome biases all local transition probabilities and makes them consistent with the selected outcome. This circular self-consistency leads to models that are inconsistent with physical reality. It is also the reason why models built solely on macroscopic observations are prone to this fallacy.

Single-molecule modeling of mRNA degradation by miRNA: Lessons from data.

Recent experimental results on the effect of miRNA on the decay of its target mRNA have been analyzed against a previously hypothesized single molecule degradation pathway. According to that hypothesis, the silencing complex (miRISC) first interacts with its target mRNA and then recruits the protein complexes associated with NOT1 and PAN3 to trigger deadenylation (and subsequent degradation) of the target mRNA. Our analysis of the experimental decay patterns allowed us to refine the structure of the degradation pathways at the single molecule level. Surprisingly, we found that if the previously hypothesized network was correct, only about 7% of the target mRNA would be regulated by the miRNA mechanism, which is inconsistent with the available knowledge. Based on systematic data analysis, we propose the alternative hypothesis that NOT1 interacts with miRISC before binding to the target mRNA. Moreover, we show that when miRISC binds alone to the target mRNA, the mRNA is degraded more slowly, probably through a deadenylation-independent pathway. The new biochemical pathway proposed here both fits the data and paves the way for new experimental work to identify new interactions.

Weak correlation of starch and volume in synchronized photosynthetic cells.

In cultures of unicellular algae, features of single cells, such as cellular volume and starch content, are thought to be the result of carefully balanced growth and division processes. Single-cell analyses of synchronized photoautotrophic cultures of the unicellular alga Chlamydomonas reinhardtii reveal, however, that the cellular volume and starch content are only weakly correlated. Likewise, other cell parameters, e.g., the chlorophyll content per cell, are only weakly correlated with cell size. We derive the cell size distributions at the beginning of each synchronization cycle considering growth, timing of cell division and daughter cell release, and the uneven division of cell volume. Furthermore, we investigate the link between cell volume growth and starch accumulation. This work presents evidence that, under the experimental conditions of light-dark synchronized cultures, the weak correlation between both cell features is a result of a cumulative process rather than due to asymmetric partition of biomolecules during cell division. This cumulative process necessarily limits cellular similarities within a synchronized cell population.

Stochastic kinetics on networks: when slow is fast.

Most chemical and biological processes can be viewed as reaction networks in which different pathways often compete kinetically for transformation of substrates into products. An enzymatic process is an example of such phenomena when biological catalysts create new routes for chemical reactions to proceed. It is typically assumed that the general process of product formation is governed by the pathway with the fastest kinetics at all time scales. In contrast to the expectation, here we show theoretically that at time scales sufficiently short, reactions are predominantly determined by the shortest pathway (in the number of intermediate states), regardless of the average turnover time associated with each pathway. This universal phenomenon is demonstrated by an explicit calculation for a system with two competing reversible (or irreversible) pathways. The time scales that characterize this regime and its relevance for single-molecule experimental studies are also discussed.

Pathway structure determination in complex stochastic networks with non-exponential dwell times.

Analysis of complex networks has been widely used as a powerful tool for investigating various physical, chemical, and biological processes. To understand the emergent properties of these complex systems, one of the most basic issues is to determine the structure and topology of the underlying networks. Recently, a new theoretical approach based on first-passage analysis has been developed for investigating the relationship between structure and dynamic properties for network systems with exponential dwell time distributions. However, many real phenomena involve transitions with non-exponential waiting times. We extend the first-passage method to uncover the structure of distinct pathways in complex networks with non-exponential dwell time distributions. It is found that the analysis of early time dynamics provides explicit information on the length of the pathways associated to their dynamic properties. It reveals a universal relationship that we have condensed in one general equation, which relates the number of intermediate states on the shortest path to the early time behavior of the first-passage distributions. Our theoretical predictions are confirmed by extensive Monte Carlo simulations.

Unveiling the hidden structure of complex stochastic biochemical networks.

Complex Markov models are widely used and powerful predictive tools to analyze stochastic biochemical processes. However, when the network of states is unknown, it is necessary to extract information from the data to partially build the network and estimate the values of the rates. The short-time behavior of the first-passage time distributions between two states in linear chains has been shown recently to behave as a power of time with an exponent equal to the number of intermediate states. For a general Markov model we derive the complete Taylor expansion of the first-passage time distribution between two arbitrary states. By combining algebraic methods and graph theory approaches it is shown that the first term of the Taylor expansion is determined by the shortest path from the initial state to the final state. When this path is unique, we prove that the coefficient of the first term can be written in terms of the product of the transition rates along the path. It is argued that the application of our results to first-return times may be used to estimate the dependence of rates on external parameters in experimentally measured time distributions.