Many aspects of the biochemistry and physiology of living cells have in the past been simulated by networks of reactions as though they were electronic circuits. In such studies, components such as receptors, enzymes, or metabolites are portrayed as being wired together in a spatially defined manner through enzymatic and other reactions. But it is clear that living circuitry is not like this; it has unique features such as a highly malleable internal architecture and the existence of a multitude of molecular states that differ in fundamental respects from those of silicon devices. Moreover, the wiring of the cell depends on the diffusive movement of myriad different molecules large and small through the watery interstices of the cytoplasm. In order to understand such systems, we need experimental techniques that can identify individual molecules and track their locations and movements within the cell. Moreover, once data of this kind are obtained we will need advanced computational methods by which spatial locations and diffusive movements of individual molecules can be represented.We recently developed a computer program for the study of intracellular reactions that allows us to take into account both the spatial location of proteins and protein complexes and their diffusive movements (1). This program uses an approach known as Brownian dynamics, in which molecules are treated as individuals rather than as concentrations and space is treated continuously instead of being subdivided into finite elements (10). Our program is called Smoldyn, for Smoluchowski dynamics, because it is based on a theory for the diffusive encounter of molecules in solution developed by the Polish physicist Marjan Smoluchowski (27). Since molecules are treated as individuals, this program can accurately capture stochastic behavior and also simulate diffusive gradients naturally and accurately. We were also inspired in our work by the MCell program, which has been developed to account for ionic and molecular events occurring within neuromuscular synapses (32). However, Smoldyn has certain advantages over MCell for our purposes since it allows reactions to occur between diffusing molecules in solution (in the current version of MCell, reactions occur only at membrane surfaces). Moreover, the code of Smoldyn, unlike that of MCell, is publicly available (http://sahara.lbl.gov/ϳsandrews/software.html).In this paper, we describe the application of Smoldyn to the well-characterized phenomenon of bacterial chemotaxis in Escherichia coli. We first predict the activity of the cluster of chemotactic receptors at one end of the cell in response to different stimulus conditions by using previously described software. We then use the temporal activity profile created in this way as an input to Smoldyn to calculate the locations of the diffusing molecules CheY, CheYp, and CheZ within the cell. As a first demonstration of the capabilities of this program, we report a series of simulations in which the diffusion of the signaling protein CheYp is followe...
Our results display the potential use of computer-based bacteria as experimental objects for exploring subtleties of chemotactic behavior.
How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.DOI: http://dx.doi.org/10.7554/eLife.00668.001
The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.The Tc1/mariner superfamily of transposons consists of the Tc1 and mariner families of elements in eukaryotes and the more distantly related IS630-like elements in bacteria (25, 39). Members of the superfamily have a single transposase gene expressed in the germ line and/or the soma, transpose via a "cut and paste" DNA intermediate, and duplicate a TA dinucleotide upon insertion. This is probably the most widespread family of transposons in nature: members have been identified in bacteria (IS630), ciliates, fungi, plants, and most animal phyla, from Porifera (sponges) to humans. Although mariner elements are widespread, they are unevenly distributed in closely related species and the vast majority are inactive because of mutations. This suggests that they have an unusual life style that involves a high rate of horizontal transfer to new hosts, followed by a burst of transposition and subsequent vertical inactivation (37, 43).Homology-dependent gene silencing serves to control the spread of transposons, retroviruses, and other repetitive DNA elements in many eukaryotes. It is mediated by at least three distinct mechanisms, specifically DNA methylation, histone deacetylation, and RNA interference. However, depending on the identity of the organism and the repetitive element in question, these mechanisms are not always completely effective. As noted by McClintock, the rate of transposition in a given germ line can change over time, with cycles of activation and silencing lasting several generations (cited in reference 35). Furthermore, although all three mechanisms of homologydependent gene silencing operate in plants and are active against many retrotransposons, som...
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