Biomedical subjects

Szabolcs Semsey

Publications and source records attributed to Szabolcs Semsey.

11 recordsLinked to original sources

PubMed2006

Genetic regulation of fluxes: iron homeostasis of Escherichia coli.

Iron is an essential trace-element for most organisms. However, because high concentration of free intracellular iron is cytotoxic, cells have developed complex regulatory networks that keep free intracellular iron concentration at optimal range, allowing the incorporation of the metal into iron-using enzymes and minimizing damage to the cell. We built a mathematical model of the network that controls iron uptake and usage in the bacterium Escherichia coli to explore the dynamics of iron flow. We simulate the effect of sudden decrease or increase in the extracellular iron level on intracellular iron distribution. Based on the results of simulations we discuss the possible roles of the small RNA RyhB and the Fe-S cluster assembly systems in the optimal redistribution of iron flows. We suggest that Fe-S cluster assembly is crucial to prevent the accumulation of toxic levels of free intracellular iron when the environment suddenly becomes iron rich.

Biological Transport↗

PubMed2006

Structure and function of negative feedback loops at the interface of genetic and metabolic networks.

The molecular network in an organism consists of transcription/translation regulation, protein-protein interactions/modifications and a metabolic network, together forming a system that allows the cell to respond sensibly to the multiple signal molecules that exist in its environment. A key part of this overall system of molecular regulation is therefore the interface between the genetic and the metabolic network. A motif that occurs very often at this interface is a negative feedback loop used to regulate the level of the signal molecules. In this work we use mathematical models to investigate the steady state and dynamical behaviour of different negative feedback loops. We show, in particular, that feedback loops where the signal molecule does not cause the dissociation of the transcription factor from the DNA respond faster than loops where the molecule acts by sequestering transcription factors off the DNA. We use three examples, the bet, mer and lac systems in Escherichia coli, to illustrate the behaviour of such feedback loops.

Down-Regulation↗

PubMed2006

Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA.

The gal operon of Escherichia coli is negatively regulated by the Gal repressosome, a higher order nucleoprotein complex containing a DNA loop that encompasses two gal promoters. In the repressosome structure, Gal repressor (GalR) dimers are bound to the two operator sites, flanking the promoter region, thus generating a DNA loop. The DNA loop is stabilized by binding of the architectural HU protein to the apex of the loop, and negative supercoiling. The gal promoters are also regulated in opposite directions by GalR without DNA looping. The repressosome-mediated as well as looping-independent transcription regulation of the two promoters is lifted in the presence of the inducer D-galactose. We tested the effect of D-galactose on various DNA-protein and protein-protein interactions of different regulatory complexes and on transcription repression in vitro. We found that the inducer breaks up the repressosome with clear intermediates in a concentration-dependent manner. The sequential disassembly generates different stages of regulation of the gal operon. The D-galactose-dependent switch from one stage of regulation to another satisfies the amphibolic requirement of the gal operon.

Binding Sites↗

PubMed2005

A gamut of loops: meandering DNA.

Nucleoprotein complexes comprising short DNA loops (150 base pairs or less) are involved in a wide variety of DNA transactions (e.g. transcription regulation, replication and recombination) in both prokaryotes and eukaryotes, and also can be useful in designing nanostructures. In these higher-order nucleoprotein complexes, proteins bound to spatially separated sites on a DNA interact with each other by looping out the relatively stiff intervening DNA. Recent technological developments have enabled determination of DNA trajectories in a few DNA-loop-containing regulatory complexes. Results show that, in a given system, a specific DNA trajectory is preferred over others.

Bacterial Proteins↗

PubMed2004

GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant.

GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.

Base Sequence↗

PubMed2004

DNA trajectory in the Gal repressosome.

The Gal repressosome is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During the repressosome assembly, a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different DNA trajectories in the repressosome. Feasibilities of these trajectories were tested by in vitro transcription repression assays, first by isolating GalR mutants with altered operator specificity and then by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four putative trajectories. Results show that OE and OI adopt a mutual antiparallel orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.

Bacterial Proteins↗

PubMed2004

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA.

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16-3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16-3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co-evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.

Agrobacterium tumefaciens↗

PubMed2003

"Antiparallel" DNA loop in gal repressosome visualized by atomic force microscopy.

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

Bacterial Proteins↗

PubMed2003

immX immunity region of rhizobium phage 16-3: two overlapping cistrons of repressor function.

16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions. We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V). The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the immX region. Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens. The X(V) part (1 kb) contained a target for X(U/L) repressor action. Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However, X(V1) could also be folded into a transcription terminator. Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.

Bacteriophages↗

PubMed2002

Operator-bound GalR dimers close DNA loops by direct interaction: tetramerization and inducer binding.

The assembly of the Gal repressosome, a higher order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli, involves the formation of a DNA loop encompassing the promoter segment. GalR dimers bound to two spatially separated operators, O(E) and O(I), specifically interact with the histone-like protein HU and close the loop in supercoiled DNA. We isolated and characterized a GalR mutant containing an amino acid substitution (R282L) that can repress transcription in the absence of HU and supercoiled DNA both in vivo and in vitro. Repression involves the same DNA looping; deletion of either O(E) or O(I) makes the mutant GalR ineffective in repression. This and other results suggest that the R282L substitution increases the normal affinity between two DNA-bound GalR dimers, allowing looping. We conclude that GalR dimers interact directly and do not use HU as an adaptor in loop closure; HU and DNA supercoiling act in concert to stabilize the GalR tetramer. The stronger GalR-GalR interaction also made the gal transcription non-inducible, suggesting that the inducer binding acts by modulating tetramerization.

DNA, Bacterial↗

PubMed2002

Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera.

The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.

Bacteriophages↗