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List of Journals

Cell

• Substrate Competition as a Source of Ultrasensitivity in the Inactivation of Wee1

Sun Young Kim1 and James E. Ferrell, Jr.

The mitotic regulators Wee1 and Cdk1 can inactivate each other through inhibitory phosphorylations. This double-negative feedback loop is part of a bistable trigger that makes the transition into mitosis abrupt and decisive. To generate a bistable response, some component of a double-negative feedback loop must exhibit an ultrasensitive response to its upstream regulator. Here, we experimentally demonstrate that Wee1 exhibits a highly ultrasensitive response to Cdk1. Several mechanisms can, in principle, give rise to ultrasensitivity, including zero-order effects, multisite phosphorylation, and competition mechanisms. We found that the ultrasensitivity in the inactivation of Wee1 arises mainly through two competition mechanisms: competition between two sets of phosphorylation sites in Wee1 and between Wee1 and other high-affinity Cdk1 targets. Based on these findings, we were able to reconstitute a highly ultrasensitive Wee1 response with purified components. Competition provides a simple way of generating the equivalent of a highly cooperative allosteric response. [1]

Molecular Microbiology

• The molecular basis of selective promoter activation by the σS subunit of RNA polymerase (REVIEW)

Athanasios Typas, Gisela Becker and Regine Hengge

Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, σS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, σ70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing σS or σ70, or by both holoenzymes. How these promoter features impose σS selectivity is further discussed. Moreover, additional pathways allow σS to compete more efficiently than σ70 for limiting amounts of core RNAP (E) and thereby enhance EσS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes. [2]

PNAS

• Combinatorial transcriptional control of the lactose operon of Escherichia coli

Thomas Kuhlman*, Zhongge Zhang, Milton H. Saier, Jr., and Terence Hwa*

The goal of systems biology is to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents involved in a biological system and their complex web of interactions. The lac promoter of Escherichia coli offers the possibility of confronting "system-level" properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offer insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators Lac repressor (LacR) and cAMP-receptor protein (CRP). A previous in vivo study [Setty Y, Mayo AE, Surette MG, Alon U (2003) Proc Natl Acad Sci USA 100:7702–7707] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors that significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we were able to account for the combinatorial control of the lac promoter quantitatively, in terms of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP. [3]

• Pharmacological ascorbic acid concentrations selectively kill cancer cells through all or none protein segregation at mitosis

Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, Shacter E, Levine M.

Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have a novel role in cancer treatment. Here we show that ascorbate affects the randomness of protein segregation at the mitosis of cancer cells, an effect that is enhanced in the presence of H2O2. We select 10 proteins from different organelles which are overexpressed in a cancer cell, Human Burkitt’s lymphoma cells (JLP-119), and tag them with EYFP to quantify the segregation error at cell division. Under the high dosage of ascorbate and H2O2 all of these proteins and corresponding organelles are lumped into only one daughter cell, leaving one cell over-populated and the other cell empty which leads to apoptosis for both of the daughter cells. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC50 values of <4 mM, a concentration easily achievable in i.v. To describe in more detail the mechanism of ascorbate-impaired partitioning, we create a general analytic description of the partition statistics. Based on the fluctuation-dissipation theorem, we also suggest a stochastic model of protein segregation at cell division which accounts for proteins in the cytoplasm and those in organelles. The model applies to both normal cells and ascorbate-treated cells. This analytical result thus explains the experimental observations. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for the role of asymmetric segregation in the treatment of infections. [4]

PRLandE

Stochastic simulations of genetic switch systems

Adiel Loinger,1 Azi Lipshtat,2 Nathalie Q. Balaban,1 and Ofer Biham1

Genetic switch systems with mutual repression of two transcription factors are studied using deterministic methods (rate equations) and stochastic methods (the master equation and Monte Carlo simulations). These systems exhibit bistability, namely two stable states such that spontaneous transitions between them are rare. Induced transitions may take place as a result of an external stimulus. We study several variants of the genetic switch and examine the effects of cooperative binding, exclusive binding, protein-protein interactions, and degradation of bound repressors. We identify the range of parameters in which bistability takes place, enabling the system to function as a switch. Numerous studies have concluded that cooperative binding is a necessary condition for the emergence of bistability in these systems. We show that a suitable combination of network structure and stochastic effects gives rise to bistability even without cooperative binding. The average time between spontaneous transitions is evaluated as a function of the biological parameters.

Stochastic suppression of gene expression oscillators under intercell coupling

A. Koseska,1 A. Zaikin,1,2 J. García-Ojalvo,3 and J. Kurths1

This paper examines the dynamics of an ensemble of hysteresis-based genetic relaxation oscillators, focusing on the influence of noise and cell-to-cell coupling on the appearance of new dynamical regimes. In particular, we show that control of the coupling strength and noise can effectively change the dynamics of the system leading to behaviors such as clustering, synchronous and asynchronous oscillations, and suppression. Moreover, under certain conditions an optimal amount of noise can lead to increased order in the system. The results obtained are correlated with relevant biological processes that occur in living organisms.

Science

Science 23 March 2007: Vol. 315. no. 5819, pp. 1716 - 1719

Tunability and Noise Dependence in Differentiation Dynamics
Gürol M. Süel,1 Rajan P. Kulkarni,2 Jonathan Dworkin,3 Jordi Garcia-Ojalvo,4 Michael B. Elowitz2*

The dynamic process of differentiation depends on the architecture, quantitative parameters, and noise of underlying genetic circuits. However, it remains unclear how these elements combine to control cellular behavior. We analyzed the probabilistic and transient differentiation of Bacillus subtilis cells into the state of competence. A few key parameters independently tuned the frequency of initiation and the duration of competence episodes and allowed the circuit to access different dynamic regimes, including oscillation. Altering circuit architecture showed that the duration of competence events can be made more precise. We used an experimental method to reduce global cellular noise and showed that noise levels are correlated with frequency of differentiation events. Together, the data reveal a noise-dependent circuit that is remarkably resilient and tunable in terms of its dynamic behavior.

Systems Biology

• Limitations of the dual reporter method and how to fix it

Longo D and Hasty J

One of the main and naturally appealing tasks of systems biology is system's dissection and identification: knowing who does what to whom and when. A unifying theme of many recent studies has been a focus on the development and utilization of single-cell experimental techniques that allow to distinguish the sources of noise associated with many cellular processes. Recently, novel information about gene expression dynamics has been obtained from single-cell experiments that draw upon the unique capabilities of using a pair fluorescent reporter proteins. In this article we present a combination of experimental and theoretical findings that illustrate the limitations of the published dual reporter method and how it can be extended to reliably infer the sources of noise in any given cellular system. We expect the results presented herein to form the backbone of all future studies on noise in cellular processes. [5]