Alexander Ninfa - Academia.edu (original) (raw)

Papers by Alexander Ninfa

Biochemistry, 2012

Uridylyltransferase/uridylyl-removing enzyme (UTase/UR) catalyzes uridylylation of PII and deurid... more Uridylyltransferase/uridylyl-removing enzyme (UTase/UR) catalyzes uridylylation of PII and deuridylylation of PII-UMP, with both activities regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing wild-type UTase/UR, the steady-state modification of PII varied from nearly complete modification to nearly complete demodification as glutamine was varied, whether the level of PII was saturating or unsaturating, but when a His-tagged version of UTase/UR was used, the robustness to variations in PII concentration was lost and the range of PII modification states in response to glutamine became smaller as the PII concentration increased. The presence of the His tag on UTase/UR did not alter PII substrate inhibition of the UT activity and had little effect on the level of the UT activity but resulted in a slight defect in UR activity. Importantly, at high PII concentrations, glutamine inhibition of the UT activity was incomplete. We hypothesized that binding of PII to the UR active site in the HD domain was responsible for PII substrate inhibition of the UT activity and, in the His-tagged enzyme, also weakened glutamine inhibition of the UT activity. Consistent with this, three different UTase/UR proteins with HD domain alterations lacked substrate inhibition of UT activity by PII; in one case, the HD alteration eliminated glutamine regulation of UT activity, while for the other two proteins, alterations of the HD domain partially compensated for the effect of the His tag in restoring glutamine regulation of UT activity. We conclude that very strong inhibition of UT activity was required for the UTase/UR-PII cycle to display robustness to the PII concentration, that in the wild-type enzyme PII brings about substrate inhibition of the UT activity by binding to the HD domain of the enzyme, and that addition of an N-terminal His tag resulted in an altered enzyme with subtle changes in the interactions between domains such that binding of PII to the HD domain interfered with glutamine regulation of the UT domain.

Microbiology and Molecular …, 1989

JEFFRY B. STOCK,* ALEXANDER J. NINFA,t AND ANN M. STOCK ... Departments ofBiology and Chemistry, ... more JEFFRY B. STOCK,* ALEXANDER J. NINFA,t AND ANN M. STOCK ... Departments ofBiology and Chemistry, Princeton University, Princeton, ... Signal Transduction Pathways That Regulate PhoB .......................................... 467 ... Adaptive responses in bacteria range from rapid ...

Biochemistry, 2009

ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adeny... more ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adenylylation and deadenylylation of GS in response to signals of cellular nitrogen status and energy. The adenylyltransferase (AT) activity of ATase is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by PII-UMP. Conversely, the adenylyl-removing (AR) activity of ATase is activated by PII-UMP and inhibited by unmodified PII and by glutamine. Here, we show that the enzyme can be reconstituted from two purified polypeptides that comprise the N-terminal two-thirds of the protein and the C-terminal one-third of the protein. Properties of the reconstituted enzyme support recent hypotheses for the sites of regulatory interactions and mechanisms for intramolecular signal transduction. Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain. Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains. Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

The homotrimeric PII signal transduction protein of Escherichia coli interacts with two small-mol... more The homotrimeric PII signal transduction protein of Escherichia coli interacts with two small-molecule effectors, 2-ketoglutarate and ATP, regulates two protein receptors, the kinase/phosphatase nitrogen regulator II (NRII) and the glutamine synthetase (GS) adenylyltransferase (ATase), and is subject to reversible uridylylation, catalyzed by the uridylyltransferase/uridylyl-removing enzyme (UTase/UR). The site of PII uridylylation, Y51, is located at the apex of the solvent-exposed T-loop (E. Cheah, P. D. Carr, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Structure 2:981-990, 1994), and an internally truncated PII lacking residues 47 to 53 formed trimers that bound the small-molecule effectors but were unable to be uridylylated or activate NRII and ATase (P. 179:4342-4353, 1997). We investigated the ability of heterotrimers containing ⌬47-53 and wild-type subunits to become uridylylated and activate NRII and ATase. Heterotrimers were formed by denaturation and renaturation of protein mixtures; when such mixtures contained a fivefold excess of ⌬47-53 subunits, the wild-type subunits were mostly redistributed into trimers containing one wild-type subunit and two mutant subunits. The resulting population of trimers was uridylylated and deuridylylated by UTase/UR, stimulated the phosphatase activity of NRII, and stimulated adenylylation of GS by ATase. In all except the ATase interaction, the activity of the hybrid trimers was greater than expected based on the number of wild-type subunits present. These results indicate that a single T-loop region within a trimer is sufficient for the productive interaction of PII with its protein receptors. We also formed heterotrimers containing wild-type subunits and subunits containing the G89A alteration (P. 179: 4342-4353, 1997). The G89A mutant form of PII does not bind the small-molecule effectors, does not interact with UTase or with NRII, and interacts poorly with ATase. Heterotrimers formed with a 10/1 starting ratio of G89A to wild-type subunits interacted with UTase/UR and ATase to a lesser extent than expected based on the number of wild-type subunits present but activated NRII slightly better than expected based on the number of wild-type subunits present. Thus, intersubunit interactions within the PII trimer can adversely affect the activity of wild-type subunits and may affect the interactions with the different receptors in a variable way. Finally, we formed heterotrimers containing ⌬47-53 and G89A mutant subunits. These heterotrimers were not uridylylated, did not interact with NRII, and interacted with the ATase only to the extent expected based on the number of G89A subunits present. Thus, the G89A subunits, which contain an intact T-loop region, were not "repaired" by inclusion in heterotrimers along with ⌬47-53 subunits.

Journal of bacteriology, 1999

The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regu... more The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in Escherichia coli. The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated. The phosphatase activity of NRII is activated by the PII signal transduction protein. We showed that PII was also an inhibitor of the kinase activity of NRII. The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated. The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII. We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleava...

Journal of bacteriology, 1997

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing... more The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudeva...

Proceedings of The National Academy of Sciences, 2001

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 ... more Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Room 5123, Detroit, MI 48201 Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved October 22, 2001 (received for review September 10, 2001) Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of

Methods in Enzymology, 2003

Regulation of expression of eukaryotic genes depends almost entirely on enhancers30-to 200-bp DN... more Regulation of expression of eukaryotic genes depends almost entirely on enhancers30-to 200-bp DNA sequences usually composed of several binding sites for an activator protein(s). The landmark of enhancers is their ability to activate target genes over a large ...

Current Topics in Cellular Regulation, 2001

... CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 36 Integration of Antagonistic Signals in the R... more ... CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 36 Integration of Antagonistic Signals in the Regulation of Nitrogen Assimilation in Escherichia coli ALEXANDER J. NINFA PENG JIANG MARIETTE R. ATKINSON JAMES A. PELISKA Department of Biological ...

Two‐Component Signaling Systems, Part A, 2007

Synthetic biology is an emerging field in which the procedures and methods of engineering are ext... more Synthetic biology is an emerging field in which the procedures and methods of engineering are extended to living organisms, with the long-term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment, or effect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable, parts, that can be introduced into cells and results in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where inpendent software modules can be independently developed and later combined into the final program.

Science Signaling, 2011

This information is current as of 12 October 2011.

2008 American Control Conference, 2008

In standard control systems theory, a system is modeled as an input/output (I/O) module with inte... more In standard control systems theory, a system is modeled as an input/output (I/O) module with internal dynamics. This implicitly assumes that the dynamics of a module do not change when the module is connected to other components. However, such an assumption is not realistic in a wide number of electrical, hydraulic, mechanical, and biological systems. We thus propose a new model that incorporates signals that travel from downstream to upstream, which we broadly call retroactivity. We quantify such a retroactivity in transcriptional components and show how to attenuate its effect by the design of insulation devices.

Science Signaling, 2012

ABSTRACT Straube suggests that a model that reflects the bifunctional nature of the cycle enzyme ... more ABSTRACT Straube suggests that a model that reflects the bifunctional nature of the cycle enzyme uridylyltransferase/uridylyl-removing enzyme (UTase/UR) should be used, in which the UT and UR activities are distinct and reciprocally regulated activity states of the enzyme, and notes that if such a model is used, the effects of retroactivity at intermediate stimulation will be different. However, such a model does not accurately match the observed enzyme regulatory properties and fails to predict the ultrasensitive response obtained in the experiments. Here, we argue that modeling the UTase/UR enzyme as a bifunctional enzyme with reciprocally regulated activity states misses important aspects of the system.

Proceedings of the National Academy of Sciences, 2001

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distan... more Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.

Proceedings of the National Academy of Sciences, 1988

We demonstrate by using purified bacterial components that the protein kinases that regulate chem... more We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated ginA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.

Nucleic Acids Research, 2002

The mechanism by which an enhancer activates transcription over large distances has been investig... more The mechanism by which an enhancer activates transcription over large distances has been investigated. Activation of the glnAp2 promoter by the NtrCdependent enhancer in Escherichia coli was analyzed using a purified system supporting multiple-round transcription in vitro. Our results suggest that the enhancer-promoter interaction and the initiation complex must be formed de novo during every round of transcription. No protein remained bound to the promoter after RNA polymerase escaped into elongation. Furthermore, the rate of initiation during the first and subsequent rounds of transcription were very similar, suggesting that there was no functional 'memory' facilitating multiple rounds of transcription. These studies exclude the hypothesis that enhancer action during multiple-round transcription involves the memory of the initial activation event.

Molecular Systems Biology, 2008

Modularity plays a fundamental role in the prediction of the behavior of a system from the behavi... more 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.

Journal of Biological Chemistry, 1996

The histidine protein kinase CheA plays an essential role in stimulus-response coupling during ba... more The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a ␥-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA 2 7 2CheA; K D ‫؍‬ 0.2-0.4 M) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (K D values Ϸ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheAϳP (K D Ϸ 0.8 mM) compared with ADP (K D Ϸ 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (k cat Ϸ 10 min ؊1 ).

Journal of Bacteriology, 2002

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by ... more The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRIϳP (NtrCϳP). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have lessefficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Developmental Biology, 2006