Synthesis Of pppGpp Research Articles

Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1–4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

TRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis.

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra- and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response.

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.

The Flexible N-terminal Domain of Ribosomal Protein L11 from Escherichia coli Is Necessary for the Activation of Stringent Factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1–72) connected to an RNA-binding C-terminal domain (amino acid residues 76–142) by a flexible linker (amino acid residues 73–75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(ΔL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

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.

Replacement of pseudouridine in transfer RNA by 5-fluorouridine does not affect the ability to stimulate the synthesis of guanosine 5'-triphosphate 3'-diphosphate

The requirement for ribothymidine and pseudouridine in the TpsiCG loop of tRNA for its activity in the ribosome and tRNA-stimulated synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) by stringent factor has been tested by the use of a purified tRNAPhe (883 pmol of phenylalanine incorporated/A260 unit) in which 92% of the pseudouridine, 98% of the ribothymidine, 98% of the dihydrouridine, and 88% of the uridines were substituted by 5-fluorouridine. This tRNA was quantitatively as active as control tRNA in inducing pppGpp synthesis. With loose-couple ribosomes, the concentration of tRNA needed to give half-maximal reaction was 0.07 micrometer for both normal and fluorouridine-substituted tRNA, with vacant tight-couple ribosomes it was 0.05 micrometer, and with tight couples carrying poly(Phe)-tRNA at the P site the value was 0.02 micrometer. These results show that at the level of intact tRNA there is no special requirement for modified bases in the TpsiCG loop of tRNA in the synthesis of pppGpp.

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.

PpGpp cycle in Escherichia coli

Kinetics of accumulation and degradation of ppGpp and pppGpp were analysed in spoT+ and spoT strains of Escherichia coli. The experimental data in this paper indicate that on degradation ppGpp is not converted to pppGpp but instead is converted to GDP which is in turn phosphorylated to GTP. In addition the data are consistent with the idea the pppGpp is a direct precursor of ppGpp. We propose that ppGpp is metabolised according to the following pathway: GTP-pppGpp--ppGpp--GDP--GTP, which we call the ppGpp cycle. Coupled with the observations in spot strains we assume that ppGpp blocks its own synthesis by inhibiting the synthesis of pppGpp but not the interconversion of the two nucleotides.

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Mutants in the spo T gene have been isolated as stringent second site revertants of the relC mutation. These show varying degrees of the characteristics associated with the spoT1 gene, viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of 3H-guanosine into GTP and ppGpr pools in spoT+ and spoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower in spoT- than in spoT+ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower in spoT than in spoT+ cells. In one of the "intermediate" spoT mutants the rate of entry of 3H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is: GDP leads to GTP leads to pppGpp leads to ppGpp leads to Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in various spoT+ and spoT- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis.

Stringent factor-independent synthesis of pppGpp in Escherichia coli strains

Stringent factor-independent synthesis of pppGpp in Escherichia coli strains

Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA.

Uncharged tRNA is preferentially bound to the peptidyl site of the ribosome in the absence of stringent factor ,but in its presence is directed to the acceptor site. The synthesis of pppGpp and ppGpp is initiated by tRNA bound in the acceptor but not in the peptidyl site. In this reaction, tRNA is not permanently attached to the acceptor site. Uncharged [32P] tRNA but not 3H-labeled stringent factor is released from the ribosome after each round of stringent factor-dependent hydrolysis of ATP. ATP-32PPi-exchange experiments reveal that exchange is independent of the presence of GTP but strongly enhanced by the addition of stringent factor and tRNA. The tRNA release is suppressed when ATP is replaced by beta, gamma-imido adenosine 5'-triphosphate, 5'-AMP, or GTP.

Studies on the in vitro synthesis of ppGpp and pppGpp

The effect of a number of inhibitors of protein synthesis on ppGpp and pppGpp synthesis in vitro has been examined. As expected from in vivo results, chloramphenicol is without effect on this reaction. Aurintricarboxylic acid and chlortetracycline, on the other hand rapidly and specifically inhibit ppGpp synthesis. Fusidic acid in the presence of saturating amounts of EF G also inhibits the reaction completely, suggesting that an empty ribosomal acceptor site is necessary for this reaction. On the other hand, the 50-S subunit proteins L7 and L12 are not required for stringent factor activity. Ribosomes from Pseudomonas fluorescens can replace those from Escherichia coli in the complete system, while ribosomes from Ehrlich ascites cannot. A small but reproducible synthesis of ppGpp is observed when the ribosomal wash from E. coli is complemented with ribosomes from wheat germ cytoplasm.

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.

Physicochemical and enzymatic properties of ATP: Nucleotide pyrophosphotransferase.

The molecular weight determined by the sedimentation equilibrium and SDS Polyacrylamide gel electrophoresis was 29,000 and 28,000, respectively. Isoelectric point of the enzyme was determined as pH 7.7. This enzyme contained large amounts of alanine, aspartic acid, glutamic acid and serine, and no cysteine residue was found. The enzyme was inhibited by SDS, KMnO4, EDTA and tetracycline. GTP and GDP were the most active as pyrophosphate acceptor to the enzyme. The apparent Km for ATP was 2.2×10−4 m and that for GTP was 2.1×10−4m in the reaction of ATP+GTP→AMP+pppGpp. On the other hand, in the reaction of 2ATP→AMP+pppApp, the apparent Km for donor and acceptor ATP was 1.7×10−3m. Effects of pH and metal ions on the enzymatic synthesis of pppGpp were also studied.

Isolation and Properties of a Ribosome-bound Factor Required for ppGpp and pppGpp Synthesis in Escherichia coli

Abstract A scheme for isolating the stringent factor, a factor which stimulates the pyrophosphorylation of GTP and GDP, from high salt extracts of ribosomes derived from stringent strains of Escherichia coli is reported. The stringent factor was purified 400-fold from the high salt-ribosome eluates. The final preparation was judged to be 85% homogeneous by sedimentation and electrophoretic criteria. The results of sodium dodecyl sulfate-gel ionophoresis and sedimentation analysis indicated that the factor is a protein consisting of a single polypeptide chain with a molecular weight of 77,000. In addition to the stringent factor, the conversion of GTP to the guanosine polyphosphates, ppGpp and pppGpp, was shown to require ATP, Mg2+, and ribosomes. The in vitro reaction was further characterized with respect to kinetic parameters, substrate specificity, and its sensitivity to known inhibitors of the in vivo synthesis of ppGpp. From measurements of stringent factor activity in fractions obtained by differential centrifugation of crude cell extracts it was concluded that essentially all of the factor in the extracts was bound to ribosomes. Extracts prepared from several different relaxed mutants were shown to be devoid of stringent factor activity. Evidence is presented that it is the stringent factor rather than the ribosomes which is impaired in these mutants. The implication of these results in relation to the function of the rel gene and the control mechanism underlying the stringent response is discussed.