Effects of ribosomes on the kinetics of Qβ replication

AbstractBacteriophage Qβ utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qβ replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single- and double-strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qβ replicase with ribosomes and to design amplifiable RNA genomes in translation-coupling systems

Quantifying epistatic interactions among the components constituting the protein translation system

In principle, the accumulation of knowledge regarding the molecular basis of biological systems should allow the development of large-scale kinetic models of their functions. However, the development of such models requires vast numbers of parameters, which are difficult to obtain in practice. Here, we used an in vitro translation system, consisting of 69 defined components, to quantify the epistatic interactions among changes in component concentrations through Bahadur expansion, thereby obtaining a coarse-grained model of protein synthesis activity. Analyses of the data measured using various combinations of component concentrations indicated that the contributions of larger than 2-body inter-component epistatic interactions are negligible, despite the presence of larger than 2-body physical interactions. These findings allowed the prediction of protein synthesis activity at various combinations of component concentrations from a small number of samples, the principle of which is applicable to analysis and optimization of other biological systems. Moreover, the average ratio of 2- to 1-body terms was estimated to be as small as 0.1, implying high adaptability and evolvability of the protein translation system

Importance of Parasite RNA Species Repression for Prolonged Translation-Coupled RNA Self-Replication

SummaryIncreasingly complex reactions are being constructed by bottom-up approaches with the aim of developing an artificial cell. We have been engaged in the construction of a translation-coupled replication system of genetic information from RNA and a reconstituted translation system. Here a mathematical model was established to gain a quantitative understanding of the complex reaction network. The sensitivity analysis predicted that the limiting factor for the present replication reaction was the appearance of parasitic replicators. We then confirmed experimentally that repression of such parasitic replicators by compartmentalization of the reaction in water-in-oil emulsions improved the duration of self-replication. We also found that the main source of the parasite was genomic RNA, probably by nonhomologous recombination. This result provided experimental evidence for the importance of parasite repression for the development of long-lasting genome replication systems

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction*S⃞

Protein synthesis is one of the most important reactions in the cell. Recent experimental studies indicated that this complex reaction can be achieved with a minimum complement of 36 proteins and ribosomes by reconstituting an Escherichia coli-based in vitro translation system with these protein components highly purified on an individual basis. From the protein-protein interaction (PPI) network of E. coli proteins, these minimal protein components are known to interact physically with large numbers of proteins. However, it is unclear what fraction of E. coli proteins are linked functionally with the minimal protein synthesis system. We investigated the effects of each of the 4194 E. coli ORF products on the minimal protein synthesis system; at least 12% of the entire ORF products, a significant fraction of the gene product of E. coli, affect the activity of this system. Furthermore 34% of these functional modifiers present in the PPI network were shown by mapping to be directly linked (i.e. to interact physically) with the minimal components of the PPI network. Topological analysis of the relationships between modifiers and the minimal components in the PPI network indicated clustering of the minimal components. The modifiers showed no such clustering, indicating that the location of functional modifiers is spread across the PPI network rather than clustering close to the minimal protein components. These observations may reflect the evolutionary process of the protein synthesis system

Evolutionary Consequence of a Trade-Off between Growth and Maintenance along with Ribosomal Damages.

Microorganisms in nature are constantly subjected to a limited availability of resources and experience repeated starvation and nutrition. Therefore, microbial life may evolve for both growth fitness and sustainability. By contrast, experimental evolution, as a powerful approach to investigate microbial evolutionary strategies, often targets the increased growth fitness in controlled, steady-state conditions. Here, we address evolutionary changes balanced between growth and maintenance while taking nutritional fluctuations into account. We performed a 290-day-long evolution experiment with a histidine-requiring Escherichia coli strain that encountered repeated histidine-rich and histidine-starved conditions. The cells that experienced seven rounds of starvation and re-feed grew more sustainably under prolonged starvation but dramatically lost growth fitness under rich conditions. The improved sustainability arose from the evolved capability to use a trace amount of histidine for cell propagation. The reduced growth rate was attributed to mutations genetically disturbing the translation machinery, that is, the ribosome, ultimately slowing protein translation. This study provides the experimental demonstration of slow growth accompanied by an enhanced affinity to resources as an evolutionary adaptation to oscillated environments and verifies that it is possible to evolve for reduced growth fitness. Growth economics favored for population increase under extreme resource limitations is most likely a common survival strategy adopted by natural microbes

Adaptive Evolution of an Artificial RNA Genome to a Reduced Ribosome Environment

The reconstitution of an artificial system that has the same evolutionary ability as a living thing is a major challenge in the <i>in vitro</i> synthetic biology. In this study, we tested the adaptive evolutionary ability of an artificial RNA genome replication system, termed the translation-coupled RNA replication (TcRR) system. In a previous work, we performed a study of the long-term evolution of the genome with an excess amount of ribosome. In this study, we continued the evolution experiment in a reduced-ribosome environment and observed that the mutant genome compensated for the reduced ribosome concentration. This result demonstrated the ability of the TcRR system to adapt and may be a step toward generating living things with evolutionary ability

Effects of Compartment Size on the Kinetics of Intracompartmental Multimeric Protein Synthesis

The cell contents are encapsulated within a compartment, the volume of which is a fundamental physical parameter that may affect intracompartmental reactions. However, there have been few studies to elucidate whether and how volume changes alone can affect the reaction kinetics. It is difficult to address these questions <i>in vivo</i>, because forced cell volume changes, e.g., by osmotic inflation/deflation, globally alters the internal state. Here, we prepared artificial cell-like compartments with different volumes but with identical constituents, which is not possible with living cells, and synthesized two tetrameric enzymes, β-glucuronidase (GUS) and β-galactosidase (GAL), by cell-free protein synthesis. Tetrameric GUS but not GAL was synthesized more quickly in smaller compartments. The difference between the two was dependent on the rate-limiting step and the reaction order. The observed acceleration mechanism would be applicable to living cells as multimeric protein synthesis in a microcompartment is ubiquitous <i>in vivo</i>

Radiation exposure doses to the surgical team during hip surgery is significantly higher during lateral imaging than posteroanterior imaging: a cadaveric simulation study

Abstract Background Fluoroscopy is indispensable when determining appropriate and effective interventions in orthopedic surgery. On the other hand, there is growing concern about the health hazards of occupational radiation exposure. The aim of this cadaveric simulation study was to measure radiation exposure doses to the surgical team during hip surgery. Methods We reproduced the intraoperative setting of hip surgery using 7 fresh frozen cadavers (5 male, 2 female) to simulate patients and mannequins to simulate the surgeon, scrub nurse, and anesthesiologist. Six real-time dosimeters were mounted at sites corresponding to the optic lens, thyroid gland, chest, gonads, foot, and hand on each mannequin. The radiation exposure dose to each team member was measured during posteroanterior and lateral fluoroscopic imaging. Results Radiation exposure doses to the surgeon were significantly higher during 3 min of lateral imaging than during 3 min of posteroanterior imaging at the optic lens (8.1 times higher), thyroid gland (10.3 times), chest (10.8 times), and hand (19.8 times) (p = 0.018, p = 0.018, p = 0.018, and p = 0.018, respectively). During lateral imaging, the radiation doses to the nurse were 0.16, 0.12, 0.09, 0.72, and 0.38 times those to the surgeon at the optic lens, thyroid, chest, gonads, and foot, respectively. The radiation dose to the anesthesiologist was zero at all anatomic sites during posteroanterior imaging and very small during lateral imaging. Conclusions Radiation exposure dose was significantly higher during lateral imaging up to 19.8 times comparing to the posteroanterior imaging. It is effective to reduce the lateral imaging time for reducing the intraoperative radiation exposure. In addition, appropriate distance from fluoroscopy resulted in very low exposure for nurses and anesthesiologists. Surgeon should pay attention that surgical staff do not get closer than necessary to the irradiation field

Relation between growth and translation.

<p><b>A.</b> Translation activity of ribosomes. The maximal translation rates of the purified ribosomes were estimated according to GFP biosynthesis curves. The ribosomes from R0, R1 and R5 are indicated in blue, purple and green, respectively. Solid lines indicated linear regressions with the slopes of 0.064, 0.016 and 0.007 for R0, R1 and R5, respectively. The standard errors of repeated tests (n = 3–4) are indicated. <b>B.</b> Correlation between growth rate and translation rate. The translation rates at 1 nM ribosomes (data from A) are plotted against the theoretically estimated maximal growth rates <i>r</i>_<i>max</i> (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135639#pone.0135639.g003" target="_blank">Fig 3C</a>). Blue, purple and red represent R0, R1 and R5, respectively. Standard errors are indicated as described in A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135639#pone.0135639.g003" target="_blank">Fig 3C</a>. <b>C.</b> A scheme of the changes in the growth economics for maintaining cellular activity. The purple shading represents the intracellular resource (histidine) concentration balanced between incorporation (orange arrow) and consumption (green arrows). The green arrows indicate the changes in resource (histidine) redistribution for translation under poor conditions. The green shading highlights the reorganized protein translation enabling survival. The orange shading suggests the changes caused by the mutations related to membrane and transport, which required further demonstration and were excluded from the present study.</p