Malgorzata Adamczyk

Systems Biology: The elements and principles of Life

Systems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occams razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate ‘stamp collecting’, it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here.

Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.

A zinc‐finger like metal binding site in the nucleosome

UV spectroscopy demonstrated that chicken mononucleosomes bind Co(II) and Zn(II) ions at submicromolar concentrations in a tetrahedral mode, at a conserved zinc finger‐like site, composed of Cys110 and His113 residues of both H3 molecules. Neither of these metal ions substituted for another, indicating a limited binding reversibility. Molecular modeling indicated that the tetrahedral site is formed by unhindered rotations around Cα–Cβ bonds in the side chains of the zinc binding residues. The resulting local rearrangement of the protein structure shields the bound metal ion from the solvent, explaining the observed lack of reversibility of the binding. Consequences of these findings for zinc homeostasis, metal toxicology and nucleosomal regulation are discussed.

Fructose bisphosphate aldolase is involved in the control of RNA polymerase III-directed transcription

Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the C128 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fba1 is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fba1. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that the function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fba1 was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase III transcription and aldolase.

Enzyme kinetics for systems biology when, why and how.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

Revealing properties of the KfrA plasmid protein via combined DLS, AFM and electrokinetic measurements.

Physicochemical characteristics of the plasmid KfrA protein in electrolyte solutions were done using a combination of dynamic light scattering (DLS), atomic force microscopy (AFM) and electrokinetic methods. The size of the protein was determined via the diffusion coefficient measurements using DLS. It was revealed from these measurements that the protein exists in an aggregated state composed of four molecules. The size of the protein was also determined via AFM imaging of single molecules adsorbed on mica from dilute solutions at pH=3.5. It was 10.6 nm in accordance with the value predicted for an aggregate composed of four monomers in a hexagonal configuration. The aggregation number was also confirmed by kinetics measurements carried out under diffusion controlled transport using AFM imaging of proteins. Further characteristics were acquired via KfrA adsorption on polystyrene latex particles (average size of 820 nm). The electrophoretic mobility of the latex and its zeta potential were determined as a function of the coverage of the protein. The maximum monolayer coverage for pH=3.5 was 1.2 mgm(-2). Additionally, from these measurements the effective charge of KfrA tetramer equal to 12 e (elementary charges) was predicted. The KfrA monolayer on latex was used to determine the isoelectric point of the protein, which was pH=4.5. As concluded, the procedures used in our work proved advantageous for a direct determination of aggregation processes and the effective charge if minor amounts of a protein are available.

High density monolayers of plasmid protein on latex particles: experiments and theoretical modeling

Monolayers obtained by adsorption of the plasmid protein KfrA on negatively charged polystyrene latex particles under diffusion-controlled conditions at pH 3.5 were interpreted in terms of the random sequential adsorption (RSA) model. A quantitative agreement of the theoretical results derived from these calculations with experimental data was attained for the ionic strength from 0.15 up to 10−2 M. This confirmed the adsorption mechanism of KfrA molecules on latex in the form of tetramers up to 10−2 M. On the other hand, for the ionic strength of 10−3 M the experimental coverage agreed with theoretical predictions under the assumption that screening of electrostatic interaction is enhanced by the presence of counterions and negatively charged polymer chains stemming from latex particles.

Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III) driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae. In a mutant compromised in RNAP III activity there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.

Low RNA Polymerase III activity results in up regulation of HXT2 glucose transporter independently of glucose signaling and despite changing environment

Background Saccharomyces cerevisiae responds to glucose availability in the environment, inducing the expression of the low-affinity transporters and high-affinity transporters in a concentration dependent manner. This cellular decision making is controlled through finely tuned communication between multiple glucose sensing pathways including the Snf1-Mig1, Snf3/Rgt2-Rgt1 (SRR) and cAMP-PKA pathways. Results We demonstrate the first evidence that RNA Polymerase III (RNAP III) activity affects the expression of the glucose transporter HXT2 (RNA Polymerase II dependent—RNAP II) at the level of transcription. Down-regulation of RNAP III activity in an rpc128-1007 mutant results in a significant increase in HXT2 mRNA, which is considered to respond only to low extracellular glucose concentrations. HXT2 expression is induced in the mutant regardless of the growth conditions either at high glucose concentration or in the presence of a non-fermentable carbon source such as glycerol. Using chromatin immunoprecipitation (ChIP), we found an increased association of Rgt1 and Tup1 transcription factors with the highly activated HXT2 promoter in the rpc128-1007 strain. Furthermore, by measuring cellular abundance of Mth1 corepressor, we found that in rpc128-1007, HXT2 gene expression was independent from Snf3/Rgt2-Rgt1 (SRR) signaling. The Snf1 protein kinase complex, which needs to be active for the release from glucose repression, also did not appear perturbed in the mutated strain. Conclusions/Significance These findings suggest that the general activity of RNAP III can indirectly affect the RNAP II transcriptional machinery on the HXT2 promoter when cellular perception transduced via the major signaling pathways, broadly recognized as on/off switch essential to either positive or negative HXT gene regulation, remain entirely intact. Further, Rgt1/Ssn6-Tup1 complex, which has a dual function in gene transcription as a repressor-activator complex, contributes to HXT2 transcriptional activation.