Early fate of exogenous promoters in E. coli

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones

Subdiffusion of loci and cytoplasmic particles are different in compressed Escherichia coli cells

The complex physical nature of the bacterial intracellular environment remains largely unknown, and has relevance for key biochemical and biological processes of the cell. Although recent work has addressed the role of non-equilibrium sources of activity and crowding, the consequences of mechanical perturbations are relatively less explored. Here we use a microfabricated valve system to track both fluorescently labeled chromosomal loci and cytoplasmic particles in cells shortly after applying a compressive force, observing the response on time scales that are too sudden to allow for biochemical response from the cell. Cytoplasmic diffusion slows markedly on compression but the exponent governing the growth of the ensemble-averaged mean-squared displacement of cytoplasmic particles is unaffected. In contrast, the corresponding exponent for DNA loci changes significantly. These results suggest that DNA elasticity and nucleoid organization play a more important role in loci subdiffusion than cytoplasmic viscoelasticity under such short time scales

Salerno's model of DNA reanalysed: could solitons have biological significance?

We investigate the sequence-dependent behaviour of localised excitations in a toy, nonlinear model of DNA base-pair opening originally proposed by Salerno. Specifically we ask whether ``breather'' solitons could play a role in the facilitated location of promoters by RNA polymerase. In an effective potential formalism, we find excellent correlation between potential minima and {\em Escherichia coli} promoter recognition sites in the T7 bacteriophage genome. Evidence for a similar relationship between phage promoters and downstream coding regions is found and alternative reasons for links between AT richness and transcriptionally-significant sites are discussed. Consideration of the soliton energy of translocation provides a novel dynamical picture of sliding: steep potential gradients correspond to deterministic motion, while ``flat'' regions, corresponding to homogeneous AT or GC content, are governed by random, thermal motion. Finally we demonstrate an interesting equivalence between planar, breather solitons and the helical motion of a sliding protein ``particle'' about a bent DNA axis.Comment: Latex file 20 pages, 5 figures. Manuscript of paper to appear in J. Biol. Phys., accepted 02/09/0

RKappa: Statistical sampling suite for Kappa models

We present RKappa, a framework for the development and analysis of rule-based models within a mature, statistically empowered R environment. The infrastructure allows model editing, modification, parameter sampling, simulation, statistical analysis and visualisation without leaving the R environment. We demonstrate its effectiveness through its application to Global Sensitivity Analysis, exploring it in "parallel" and "concurrent" implementations. The pipeline was designed for high performance computing platforms and aims to facilitate analysis of the behaviour of large-scale systems with limited knowledge of exact mechanisms and respectively sparse availability of parameter values, and is illustrated here with two biological examples. The package is available on github: https://github.com/lptolik/R4KappaComment: Hybrid Systems and Biology 2014, Vienn

A Computational Investigation on the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins

Deciphering the universe of RNA structures and trans RNA-RNA interactions of transcriptomes in vivo: from experimental protocols to computational analyses

The last few years have seen an explosion of experimental and computational methods for investigating RNA structures of entire transcriptomes in vivo. Very recent experimental protocols now also allow trans RNA–RNA interactions to be probed in a transcriptome-wide manner. All of the experimental strategies require comprehensive computational pipelines for analysing the raw data and converting it back into actual RNA structure features or trans RNA–RNA interactions. The overall performance of these methods thus strongly depends on the experimental and the computational protocols employed. In order to get the best out of both worlds, both aspects need to be optimised simultaneously. This review introduced the methods and proposes ideas how they could be further improved

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation.

International audienceIn most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC complex and the mechanism by which it primes the origin for the initiation of replication remain unclear. In this study, we have performed magnetic tweezers experiments to investigate the structural properties of the DnaA-oriC complex. We show that the DnaA-ATP-oriC complex adopts a right-handed helical conformation involving a variable amount of DNA and protein whose features fit qualitatively as well as quantitatively with an existing model based on the crystal structure of a truncated DnaA tetramer obtained in the absence of DNA. We also investigate the topological effect of oriC's DNA unwinding element