Differential regulation of opposing RelMtb activities by the aminoacylation state of a tRNA.ribosome.mRNA.RelMtb complex

Rel(Mtb) of Mycobacterium tuberculosis is responsible for the intracellular regulation of (p)ppGpp and the consequent ability of the organism to survive long-term starvation, indicating a possible role in the pathogenesis of tuberculosis. Purified Rel(Mtb) is a dual-function enzyme carrying out ATP: GTP/GDP/ITP 3'-pyrophosphoryltransferase and (p)ppGpp 3'-pyrophosphohydrolase reactions. Here we show that in the absence of biological regulators, Rel(Mtb) simultaneously catalyzes both transferase and hydrolysis at the maximal rate for each reaction, indicating the existence of two distinct active sites. The differential regulation of the opposing activities of Rel(Mtb) is dependent on the ratio of uncharged to charged tRNA and the association of Rel(Mtb) with a complex containing tRNA, ribosomes, and mRNA. A 20-fold increase in the k(cat) and a 4-fold decrease in K(ATP) and K(GTP) from basal levels for transferase activity occur when Rel(Mtb) binds to a complex containing uncharged tRNA, ribosomes, and mRNA (Rel(Mtb) activating complex or RAC). The k(cat) for hydrolysis, however, is reduced 2-fold and K(m) for pppGpp increased 2-fold from basal levels in the presence of the Rel(Mtb) activating complex. The addition of charged tRNA to this complex has the opposite effect by inhibiting transferase activity and activating hydrolysis activity. Differential control of Rel(Mtb) gives the Mtb ribosomal complex a new regulatory role in controlling cellular metabolism in response to stringent growth conditions that may be present in the dormant Mtb lesion.     

Synthesis and hydrolysis of pppGpp in mycobacteria: a ligand mediated conformational switch in Rel

Bacteria respond to starvation by synthesizing a polyphosphate derivative of guanosine, (p)ppGpp, that helps the bacteria in surviving during stress. The protein in Gram-positive organisms required for (p)ppGpp synthesis is Rel, a bifunctional enzyme that carries out both synthesis and hydrolysis of this molecule. Rel shows increased pppGpp synthesis in the presence of uncharged tRNA, the effect of which is regulated by the C-terminal of Rel. We show by fluorescence resonance energy transfer that the distance between the N-terminus cysteine residue at the catalytic domain and C692 at the C-terminus increases upon the addition of uncharged tRNA. In apparent anomaly, the steady state anisotropy of the Rel protein decreases upon tRNA binding suggesting "compact conformation" vis-à-vis "open conformation" of the free Rel. We propose that the interaction between C692 and the residues present in the pppGpp synthesis site results in the regulated activity and this interaction is abrogated upon addition of uncharged tRNA. We also report here the binding of pppGpp to the C-terminal part of the protein that leads to more unfolding in this region.     

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.     

Functional regulation of the opposing (p)ppGpp synthetase/hydrolase activities of RelMtb from Mycobacterium tuberculosis

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.     

Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis

Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel(Seq) protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3'-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn(2+)-dependent 3'-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel(Seq) to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel(Seq) with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.     

A charge reversal differentiates (p)ppGpp synthesis by monofunctional and bifunctional Rel proteins

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.     

Activation of transcription by guanosine 5'-diphosphate,3'-diphosphate, transfer ribonucleic acid, and novel protein from Escherichia coli.

A protein factor TFms) that is required for ppGpp to stimulate RNA synthesis has been purified from an eluate of crude ribosomes. TFms also has the capacity to stimulate RNA synthesis without ppGpp present. Under standard conditions the action TFms and ppGpp requires uncharged tRNA. TFms and ppGpp act at inhibition to promote the formation of rifampicin-resistant or polytrI)-resistant preinitiation complexes. In the presence of rifampicin or poly(rI), tRNA is no longer required. With lambdah80dlacPs DNA as template, ppGpp together with TFms stimulated gal RNA synthesis to a much greater extent than total RNA synthesis. The stimulation of both lac and gel RNA synthesis was increased in the presence of cyclic AMP receptor and cyclic AMP.     

Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy

Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were the same in Rel(+) and Rel(-) strains and reduced, at most, two- to fivefold in both strains during starvation. The present reinvestigation of the charging levels of tRNA(2)(Arg), tRNA(1)(Thr), tRNA(1)(Leu) and tRNA(His) during starvation of isogenic Rel(+) and Rel(-) strains showed that starvation reduced charging levels tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging levels of tRNA(2)(Arg) and tRNA(1)(Thr) during starvation were accurate enough to demonstrate that charging levels were at least fivefold lower in the Rel(-) strain compared to the Rel(+) strain. Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome.     

Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected

Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites.     

Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.     

The stringent response of Mycobacterium tuberculosis is required for long-term survival

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.     

GTP degradation to guanine catalyzed by ribosomal subunits and microsomal-wash factors.

Ribosomes from stringent strains of bacteria generate (p)ppGpp if incubated with uncharged tRNA, a ribosomal wash fraction, GTP and ATP. By contrast, an analogous system from rat liver does not transform GTP to (p)ppGpp but degrades it to guanine. The reaction requires the ribosomal subunits, a 40 000-Mr and a 60 000-Mr microsomal wash protein factor and is inhibited if the ribosomal A-site is charged with aminoacyl tRNA. The degradation of GTP to guanine occurs in the following four distinct reaction steps: (a) hydrolysis of GTP to GDP plus Pi, (b) hydrolysis of GDP to GMP plus Pi, (c) hydrolysis of GMP to guanosine plus Pi, (d) hydrolysis of guanosine to guanine plus ribose. The reaction step (a) is inhibited by fusidic acid, cycloheximide, emetine, tetracycline and puromycin. The hydrolysis of GDP is inhibited strongly by fusidic acid, emetine and tetracycline. A putative physiological significance of this ribosome-dependent pathway in the processes of growth control of animal cells under conditions of amino acid deprivation is discussed.     

Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.     

Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis.

We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.     

Purification and properties of stringent factor.

The stringent factor from Escherichia coli is the product of the relA locus. It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP). This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation. In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e. the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates. Here we report the purification of the stringent factor to near homogeneity. It is a monomeric protein with a molecular weight of 75,000. The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.