Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input-output dynamic characteristics of transcriptional components, focusing on a property, which we call 'retroactivity', that plays a role analogous to nonzero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoterbinding sites, or when the affinity of such binding sites is high. To attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation-dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast timescales of the phosphorylation and dephosphorylation reactions.

Analysis of the system design principles of signaling systems requires model systems where all components and regulatory interactions are known. Components of the Lac and Ntr systems were used to construct genetic circuits that display toggle switch or oscillatory behavior. Both devices contain an "activator module" consisting of a modified glnA promoter with lac operators, driving the expression of the activator, NRI. Since NRI activates the glnA promoter, this creates an autoactivated circuit repressible by LacI. The oscillator contains a "repressor module" consisting of the NRI-activated glnK promoter driving LacI expression. This circuitry produced synchronous damped oscillations in turbidostat cultures, with periods much longer than the cell cycle. For the toggle switch, LacI was provided constitutively; the level of active repressor was controlled by using a lacY mutant and varying the concentration of IPTG. This circuitry provided nearly discontinuous expression of activator.

The molecular mechanisms responsible for stimulus-response coupling often involve two types of enzymatic components: histidine protein kinases (HPK), and their associated response regulators (RR). Signal transduction occurs through the transfer of phosphoryl groups from adenosine triphosphate (ATP) to histidine residues in the histidine kinases, from the HPK-phosphohistidine side chains to aspartic acid residues in the RR, and, finally, from the response regulator-phosphoaspartate side chains to water:

Transcription from nitrogen-regulated pro- NRII-independent mechanism for the regulation of transcription from glnAp2 does exist.The regulation of ginA expression by NRII requires the products of two additional genes, ginD and ginB (9, 10). The ginD gene product is a uridylyltransferase (UTase) required for the conversion of PI,, the ginB gene product, to a uridylylated form, and a uridylyl-removing enzyme, which catalyzes the reverse reaction. The ability of UTase to convert PI, to PII-UMP is stimulated by 2-ketoglutarate and, conversely, the ability of uridylyl-removing enzyme to remove the uridylyl group from PII-UMP is stimulated by glutamine (11). Thus, ammonia starvation, which results in a high intracellular ratio of 2-ketoglutarate to glutamine, causes the conversion of PI, to PII-UMP. Growth

The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia coli plays an important role in the regulation of nitrogen assimilation by controlling the uridylylation state of the PII signal transduction protein (PII) in response to intracellular signals. The reversible uridylylation of PII indirectly controls the activity of PII receptors that regulate transcription from nitrogen-regulated promoters and the activity of glutamine synthetase. Here, we present a detailed analysis of the uridylyltransferase and uridylyl-removing activities and their regulation by the small molecule effectors ATP, 2-ketoglutarate, and glutamine. Several important features of enzyme mechanism and regulation were elucidated. Mg2+ appeared to be the physiologically relevant metal ion cofactor for both transferase and uridylyl-removing activities. The transferase reaction proceeded by an ordered bi-bi kinetic mechanism, with PII binding before UTP and pyrophosphate (PPi) released before PII-UMP. The uridylyl-removing reaction proceeded with rapid equilibrium binding of substrate and random release of products. Both reactions were activated by ATP and 2-ketoglutarate, which did so by binding only to PII and PII-UMP. The binding of these effectors to PII and PII-UMP was characterized. Glutamine inhibited the transferase reaction by inhibiting the chemistry step, while glutamine provided nonessential mixed-type activation of the uridylyl-removing activity, lowering the apparent Km and increasing kcat. Our data were consistent with the hypothesis that all effects of glutamine are due to the binding of central complexes at a single glutamine site. By comparing the effects of the activators with their reported in vivo concentrations, we conclude that in intact cells the uridylylation state of PII is regulated mainly by the glutamine concentration and is largely independent of the 2-ketoglutarate concentration. Our kinetic data were consistent with the hypothesis that both transferase and uridylyl-removal reactions occurred at a single active center on the enzyme.