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.     

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.     

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.     

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.     

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.     

Molecular dissection of the mycobacterial stringent response protein Rel

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.     

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.     

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.     

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.     

Characterization of the stringent and relaxed responses of Streptococcus equisimilis.

The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.     

Evidence of a conserved intrinsically disordered region in the C-terminus of the stringent response protein Rel from mycobacteria

The RelA/SpoT enzyme produces (p)ppGpp that helps the bacterium survive during stress. The domains present in it are interspersed with connecting linkers whose functions have been poorly elucidated. We rationally analyzed the sequence and structural property of the regulatory C-terminal region in the Rel family of proteins and report the presence of an intrinsically disordered region between two successive domains in this region that are separated by a defined amino acid sequence length. We show that the length and secondary structure of this linker are conserved in Rel proteins, further signifying its importance in rendering flexibility for domain movement and domain–domain interaction.     

Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain

The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA⁺ cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.     

Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism

Bacteria respond to nutritional stresses by producing an intracellular alarmone, guanosine 5'-(tri)diphosphate, 3'-diphosphate [(p)ppGpp], which triggers the stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, upon fatty acid or carbon starvation, SpoT enzyme activity switches from (p)ppGpp degradation to (p)ppGpp synthesis, but the signal and mechanism for this response remain totally unknown. Here, we characterize for the first time a physical interaction between SpoT and acyl carrier protein (ACP) using affinity co-purifications and two-hybrid in E. coli. ACP, as a central cofactor in fatty acid synthesis, may be an ideal candidate as a mediator signalling starvation to SpoT. Accordingly, we show that the ACP/SpoT interaction is specific of SpoT and ACP functions because ACP does not interact with the homologous RelA protein and because SpoT does not interact with a non-functional ACP. Using truncated SpoT fusion proteins, we demonstrate further that ACP binds the central TGS domain of SpoT, consistent with a role in regulation. The behaviours of SpoT point mutants that do not interact with ACP reveal modifications of the balance between the two opposite SpoT catalytic activities thereby changing (p)ppGpp levels. More importantly, these mutants fail to trigger (p)ppGpp accumulation in response to fatty acid synthesis inhibition, supporting the hypothesis that the ACP/SpoT interaction may be involved in SpoT-dependent stress response. This leads us to propose a model in which ACP carries information describing the status of cellular fatty acid metabolism, which in turn can trigger the conformational switch in SpoT leading to (p)ppGpp accumulation.     

Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated 'L11) was constructed. Bacteria overexpressing 'L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed 'L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.     

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthetic activities on Escherichia coli SpoT domains

Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5'-diphosphate 3'-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, alpha-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further alpha-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA -, spoT- double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).     

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.