Functional dissection of the dimerization and enzymatic activities of Escherichia coli nitrogen regulator II and their regulation by the PII protein.

The dimeric two-component system transmitter protein NRII (NtrB) of Escherichia coli, product of glnL (ntrB), controls transcription of nitrogen-regulated genes by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI (NtrC). Previous studies showed that the PII signal transduction protein inhibits the kinase activity of NRII and activates its phosphatase activity. We observed that PII greatly stimulated the NRII phosphatase activity under conditions where the cleavage of ATP was prevented, indicating that the phosphatase activity did not result simply from prevention of the antagonistic NRII kinase activity by PII. Rather, PII was an activator of the phosphatase activity. To study this regulation, we examined the dimerization and enzymatic activities of NRII and various polypeptides derived from NRII, and their regulation by PII. Our results were consistent with the hypothesis that NRII consists of three domains: an N-terminal domain found only in NRII proteins and two domains formed by the conserved transmitter module of NRII, the phosphotransferase/phosphatase/dimerization (central) domain and the kinase domain. All three domains were involved in regulating the kinase and phosphatase activities of NRII. The N-terminal domain was involved in intramolecular signal transduction, and controlled access to the NRII active site for the isolated dimeric central domain added in trans. The central domain was responsible for dimerization and the phosphotransferase and phosphatase activities of NRII, but the latter activity was weak in the isolated domain and was not regulated by PII. The C-terminal kinase domain was responsible for the kinase activity. The PII protein appeared to interact with the isolated transmitter module of NRII, and not with the N-terminal domain as previously thought, since PII dramatically increased the stoichiometry of autophosphorylation of the isolated transmitter module. However, the phosphatase activity of the transmitter module of NRII was low even in the presence of PII, suggesting that the N-terminal domain was necessary for the central domain to assume the conformation necessary for potent phosphatase activity. Also, PII significantly reduced the rate of transphosphorylation of the isolated central domain by the isolated kinase domain, suggesting that PII interacts directly with the kinase domain. We hypothesize that the binding of PII to the kinase domain of NRII results in an altered conformation that is transmitted to the central and N-terminal domains; this causes the central domain to assume the conformation with potent phosphatase activity.

The Escherichia coli PII signal transduction protein regulates the activities of the two-component system transmitter protein NRII by direct interaction with the kinase domain of the transmitter module.

The PII signal transduction protein regulates the transcription of nitrogen-regulated genes by controlling the kinase and phosphatase activities of NRII. We used a cross-linking approach to study the interaction of the T-loop of the PII protein with NRII. Cross-linking of PII to NRII required ATP and 2-ketoglutarate, allosteric effectors known to control PII activity, and was not affected by the presence of excess nonspecific proteins such as bovine serum albumin. The purified cross-linked species appeared to consist mainly of PII trimers in which one of the three subunits was cross-linked to a single subunit of the NRII dimer; this complex had the phosphatase activity characteristic of the un-cross-linked PII-NRII complex, and had significant phosphatase activity in the absence of 2-ketoglutarate, suggesting that once PII was tethered to NRII the active conformation was stabilized. Studies with truncated forms of NRII indicated that the purified N-terminal "sensory" domain of NRII was not cross-linked to PII, nor was a polypeptide consisting of NRII residues 1-189. In contrast, polypeptides containing the kinase domain of the transmitter module of NRII (residues 190-349) were cross-linked to PII in an ATP- and 2-ketoglutarate-dependent reaction. These results indicate that PII controls NRII by interaction with the conserved kinase domain of the transmitter module.

Asymmetry in the autophosphorylation of the two-component regulatory system transmitter protein nitrogen regulator II of Escherichia coli.

Autophosphorylation of the homodimeric two-component system transmitter protein nitrogen regulator II (NRII; also NtrB) of Escherichia coli is the first step in the activation of nitrogen-regulated (Ntr) gene transcription. We show that the autophosphorylation of NRII was asymmetric, with phosphorylation of the first and second subunits of the dimer displaying different equilibria (under our experimental conditions K(1) approximately 0. 345, K(2) approximately 0.0044). Phosphorylation of both subunits of NRII was rapid, but the very rapid reversal of the phosphorylation of the second subunit was responsible for the equilibrium position of the reaction. Complete phosphorylation of NRII was only observed under conditions where ADP, a product of the autophosphorylation reaction, was removed by an enzymatic system. Purified, doubly phosphorylated NRII (NRII approximately P(2)) was stable in the absence of nucleotides at 0 degrees C but was dephosphorylated to the hemiphosphorylated form at 37 degrees C. In the presence of a low concentration of ADP, half of the phosphoryl groups from NRII approximately P(2) were rapidly dephosphorylated, while the remaining phosphoryl groups were slowly dephosphorylated. Experiments with heterodimers containing wild-type and mutant, nonphosphorylatable subunits suggested that the asymmetry of NRII autophosphorylation was not preexisting but resulted from the autophosphorylation of one subunit.

PII signal transduction proteins.

PII proteins, found in Bacteria, Archaea and plants, help coordinate carbon and nitrogen assimilation by regulating the activity of signal transduction enzymes in response to diverse signals. Recent studies of bacterial PII proteins have revealed a solution to the signal transduction problem of how to coordinate multiple receptors in response to diverse stimuli yet permit selective control of these receptors under various conditions and allow adaptation of the system as a whole to long-term stimulation.

Characterization of the GlnK protein of Escherichia coli.

The GlnK and PII signal transduction proteins are paralogues that play distinct roles in nitrogen regulation. Although cells lacking GlnK appear to have normal nitrogen regulation, in the absence of PII, the GlnK protein controls nitrogen assimilation by regulating the activities of the PII receptors glutamine synthetase adenylyltransferase (ATase) and the kinase/phosphatase nitrogen regulator II (NRII or NtrB), which controls transcription from nitrogen-regulated promoters. Here, the wild-type GlnK protein and two mutant forms of GlnK were purified, and their activities were compared with those of PII using purified components. GlnK and PII were observed to have unique properties. Both PII and GlnK were potent activators of the phosphatase activity of NRII, although PII was slightly more active. In contrast, PII was approximately 40-fold more potent than GlnK in the activation of the adenylylation of glutamine synthetase by ATase. While both GlnK and PII were readily uridylylated by the uridylyltransferase activity of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), only PII approximately UMP was effectively deuridylylated by the UR activity of the UTase/UR. Finally, there were subtle differences in the regulation of GlnK activity by the small molecule effector 2-ketoglutarate compared with the regulation of PII activity by this effector. Altogether, these results suggest that GlnK is unlikely to play a significant role in the regulation of ATase in wild-type cells, and that the main role of GlnK may be to contribute to the regulation of NRII and perhaps additional, unknown receptors in nitrogen-starved cells. Also, the slow deuridylylation of GlnK approximately UMP by the UTase/UR suggests that rapid interconversion of GlnK between uridylylated and unmodified forms is not necessary for GlnK function. One mutant form of GlnK, containing the alteration R47W, was observed to lack specifically the ability to activate the NRII phosphatase in vitro; it was able to be uridylylated by the UTase/UR and to activate the adenylylation activity of ATase. Another mutant form of GlnK, containing the Y51N alteration at the site of uridylylation, was not uridylylated by the UTase/UR and was defective in the activation of both the NRII phosphatase activity and the ATase adenylylation activity.

Regulation of autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein.

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 cleavage of ATP by NRII. The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities. Under these conditions, the kinase activity of NRII is dominant. At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity. However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII's kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).

Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein.

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.

The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed.

Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli.

Nitrogen-regulation of gene transcription in Escherichia coli results from the regulation of the phosphorylation state of the enhancer-binding transcription factor NRI (NtrC). We examined the regulation of NRI phosphorylation in a reconstituted bicyclic cascade system containing four regulatory proteins: NRI, the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate the signal transduction protein PII, and the kinase/phosphatase NRII (NtrB), which is a PII receptor that phosphorylates and dephosphorylates NRI. In this reconstituted system, the phosphorylation state of NRI was regulated reciprocally by the small molecule effectors glutamine, which prevented the accumulation of NRI-P, and 2-ketoglutarate, which caused accumulation of NRI-P. Regulation of the bicyclic system by glutamine was exclusively due to sensation and signal-transduction by the UTase/UR-PII monocycle, which was observed to function essentially as a glutamine-sensing apparatus. In contrast, regulation of NRI phosphorylation by 2-ketoglutarate, which binds to PII, was due to direct regulation of the NRII-PII interaction and the rate of NRI-P dephosphorylation. Thus, the PII protein transduces the glutamine signal to the NRII-NRI monocycle in the form of its uridylylation state and is also the receptor of the antagonistic 2-ketoglutarate signal, which blocks the activity of unmodified PII.

Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli.

Two structurally similar but functionally distinct PII-like proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphorylation state of the transcriptional activator NRI-P (NtrC-P, the phosphorylated form of the glnG (ntrC) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA, which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK*, were obtained as suppressors of the Ntr- phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRI-P during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

A protein-induced DNA bend increases the specificity of a prokaryotic enhancer-binding protein.

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase from binding sites located adjacent to the target promoter. Alternatively, in the case of genes transcribed by Escherichia coli RNA polymerase holoenzyme containing the alternate sigma factor sigma54, regulatory proteins bound at more distally located enhancer sites can activate transcription via DNA looping by taking advantage of the increasing flexibility of DNA over longer distances. While this second mechanism offers a greater possible flexibility in the location of these binding sites, it is not clear how the specificity offered by the proximity of the regulatory protein and the polymerase intrinsic to the first mechanism is maintained. Here we demonstrate that integration host factor (IHF), a protein that induces a sharp bend in DNA, acts both to inhibit DNA-looping-dependent transcriptional activation by an inappropriate enhancer-binding protein and to facilitate similar activation by an appropriate enhancer-binding protein. These opposite effects have the consequence of increasing the specificity of activation of a promoter that is susceptible to regulation by proteins bound to a distal site.

Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus.

Cell differentiation in the Caulobacter crescentus cell cycle requires differential gene expression that is regulated primarily at the transcriptional level. Until now, however, a defined in vitro transcription system for the biochemical study of developmentally regulated transcription factors had not been available in this bacterium. We report here the purification of C. crescentus RNA polymerase holoenzymes and resolution of the core RNA polymerase from holoenzymes by chromatography on single-stranded DNA cellulose. The three RNA polymerase holoenzymes Esigma54, Esigma32, and Esigma73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Esigma54 initiated transcription from the sigma54-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active Esigma32 specifically initiated transcription from the sigma32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Esigma73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length sigma73 protein. The reconstituted Esigma73 recognized the sigma70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the sigma73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Esigma73 RNA polymerase to recognize E. coli sigma70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.

Probing interactions of the homotrimeric PII signal transduction protein with its receptors by use of PII heterotrimers formed in vitro from wild-type and mutant subunits.

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. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179:4342-4353, 1997). We investigated the ability of heterotrimers containing delta47-53 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 A47-53 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. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 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 delta47-53 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 delta47-53 subunits.

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

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. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.

Identification, characterization, and chromosomal organization of cell division cycle genes in Caulobacter crescentus.

We report a detailed characterization of cell division cycle (cdc) genes in the differentiating gram-negative bacterium Caulobacter crescentus. A large set of temperature-sensitive cdc mutations was isolated after treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Analysis of independently isolated mutants at the nonpermissive temperature identified a variety of well-defined terminal phenotypes, including long filamentous cells blocked at various stages of the cell division cycle and two unusual classes of mutants with defects in both cell growth and division. The latter strains are uniformly arrested as either short bagel-shaped coils or large predivisional cells. The polar morphology of these cdc mutants supports the hypothesis that normal cell cycle progression is directly responsible for developmental regulation in C. crescentus. Genetic and physical mapping of the conditional cdc mutations and the previously characterized dna and div mutations identified at least 21 genes that are required for normal cell cycle progression. Although most of these genes are widely scattered, the genetically linked divA, divB, and divE genes were shown by genetic complementation and physical mapping to be organized in one gene cluster at 3200 units on the chromosome. DNA sequence analysis and marker rescue experiments demonstrated that divE is the C. crescentus ftsA homolog and that the ftsZ gene maps immediately adjacent to ftsA. On the basis of these results, we suggest that the C. crescentus divA-divB-divE(ftsA)-ftsZ gene cluster corresponds to the 2-min fts gene cluster of Escherichia coli.

Activation of transcription initiation from the nac promoter of Klebsiella aerogenes.

The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRI approximately P than did the activation of the Escherichia coli glnAp2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma 54 RNA polymerase than did glnAp2, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp2 promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp2 promoter was decreased at low concentrations of NRI approximately P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI approximately P binding site was strongly affected by subtle alterations in the position of the NRI approximately P binding site(s), indicating a face-of-the-helix dependency for activation.

Repression of the Klebsiella aerogenes nac promoter.

Transcription of the nitrogen-regulated nac promoter of Klebsiella aerogenes requires sigma54 RNA polymerase, is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), and is repressed by the product of the nac gene, Nac. Nac protects a large portion of the nac control region, extending from positions -130 to -70, from digestion by DNase I. This site(s) lies immediately upstream from the site at which sigma 54 RNA polymerase binds, is downstream of a high-affinity binding site for the transcriptional activator NRI approximately P, and partially overlaps a low-affinity NRI approximately P-binding site. Binding of Nac to the DNA resulted in bending of the DNA but did not interfere with the binding of sigma 54 RNA polymerase to the promoter or with the binding of NRI approximately P to either the high-affinity site or low-affinity site. Furthermore, transcription assays with various wild-type and mutant templates suggested that Nac did not exclude NRI approximately P from either the low- or high-affinity sites, nor did Nac interfere with the ability of the polymerase to form the open complex when the binding sites for NRI approximately P were moved to different locations upstream from the promoter. Rather, Nac seemed to repress by an antiactivation mechanism in which the interaction of the NRI approximately P, bound at its normal sites, with sigma 54 RNA polymerase, bound to the promoter, was prevented.

The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP.

Nitrogen regulation of transcription in Escherichia coli requires sensation of the intracellular nitrogen status and control of the dephosphorylation of the transcriptional activator NRI-P. This dephosphorylation is catalyzed by the bifunctional kinase/phosphatase NRII in the presence of the dissociable PII protein. The ability of PII to stimulate the phosphatase activity of NRII is regulated by a signal transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which converts PII to PII-UMP under conditions of nitrogen starvation; this modification prevents PII from stimulating the dephosphorylation of NRI approximately P. We used purified components to examine the binding of small molecules to PII, the effect of small molecules on the stimulation of the NRII phosphatase activity by PII, the retention of PII on immobilized NRII, and the regulation of the uridylylation of PII by the UTase/UR enzyme. Our results indicate that PII is activated upon binding ATP and either 2-ketoglutarate or glutamate, and that the liganded form of PII binds much better to immobilized NRII. We also demonstrate that the concentration of glutamine required to inhibit the uridylyltransferase activity is independent of the concentration of 2-ketoglutarate present. We hypothesize that nitrogen sensation in E. coli involves the separate measurement of glutamine by the UTase/UR protein and 2-ketoglutarate by the PII protein.

Preliminary X-ray diffraction analysis of crystals of the PII protein from Escherichia coli.

PII protein, which carries metabolic signals regulating the transcription and activity of glutamine synthetase in nitrogen assimilation in Escherichia coli, has been crystallized in space group P2(1) with a = 47.8 A, b = 62.9 A, c = 52.8 A and beta = 100.3 degrees and space group P2(1)2(1)2(1) with a = 52.2 A. b = 64.9 A and c = 100.1 A. Both the monoclinic crystals, which diffract beyond 3.0 A, and the orthorhombic crystals, which diffract beyond 2.5 A, probably have three molecules of 12,400 Da each in the crystallographic asymmetric unit.