117 research outputs found
Two distinct logical types of network control in gene expression profiles
In unicellular organisms such as bacteria the same acquired mutations
beneficial in one environment can be restrictive in another. However, evolving
Escherichia coli populations demonstrate remarkable flexibility in adaptation.
The mechanisms sustaining genetic flexibility remain unclear. In E. coli the
transcriptional regulation of gene expression involves both dedicated
regulators binding specific DNA sites with high affinity and also global
regulators - abundant DNA architectural proteins of the bacterial chromoid
binding multiple low affinity sites and thus modulating the superhelical
density of DNA. The first form of transcriptional regulation is dominantly
pairwise and specific, representing digitial control, while the second form is
(in strength and distribution) continuous, representing analog control. Here we
look at the properties of effective networks derived from significant gene
expression changes under variation of the two forms of control and find that
upon limitations of one type of control (caused e.g. by mutation of a global
DNA architectural factor) the other type can compensate for compromised
regulation. Mutations of global regulators significantly enhance the digital
control; in the presence of global DNA architectural proteins regulation is
mostly of the analog type, coupling spatially neighboring genomic loci;
together our data suggest that two logically distinct types of control are
balancing each other. By revealing two distinct logical types of control, our
approach provides basic insights into both the organizational principles of
transcriptional regulation and the mechanisms buffering genetic flexibility. We
anticipate that the general concept of distinguishing logical types of control
will apply to many complex biological networks.Comment: 19 pages, 6 figure
iSLIM: a comprehensive approach to mapping and characterizing gene regulatory networks
Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryote
Massively parallel measurements of molecular interaction kinetics on a microfluidic platform
Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)—DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2×10^(-6)M to 2×10^(-9)M, and dissociation rate constants of approximately 6s^(-1) to 8.5×10^(-3)s^(-1). Association rate constants were uniform across 3 TF families, ranging from 3.7x10^6 M^(-1)s^(-1) to 9.6x10^7 M^(-1)s^(-1), and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems
High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes
The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing
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