Guanosine pentaphosphate
Encyclopedia
ppGpp, guanosine pentaphosphate or tetraphosphate is an alarmone
which is involved in the stringent response
in bacteria
, causing the inhibition of RNA synthesis when there is a shortage of amino acid
s present. This causes translation to decrease and the amino acid
s present are therefore conserved. Furthermore, genes for amino acid uptake (transport into the cell from surrounding media) and biosynthesis are also upregulated. However, (p)ppGpp influences the overall gene expression
pattern in the cell so that many genes required for survival in stressful conditions are up-regulated. A complete absence of (p)ppGpp generates its own unique phenotypic features in E. coli. These include multiple amino acid requirements, poor survival of aged cultures, aberrant cell division, morphology, and immotility, as well as being locked in a growth mode during entry into starvation.
(p)ppGpp is created via pppGpp synthase
, also known as RelA, and is converted from pppGpp to ppGpp via pppGpp phosphohydrolase. RelA is associated with about every one in two hundred ribosome
s and it becomes activated when an uncharged transfer RNA
(tRNA) molecule enters the A site of the ribosome, due to the shortage of amino acid required by the tRNA. If a mutant
bacterium is relA- it is said to be relaxed and no regulation of RNA production due to amino acid absence is seen.
Targets of (p)ppGpp include rRNA operon
s, of which there are seven in Escherichia coli
(a commonly used bacteria
l model organism
), all of which have 2 promoters. When (p)ppGpp associates with the promoter it affects the RNA polymerase
enzyme
's ability to bind and initiate transcription
. It is thought that (p)ppGpp may affect the stability of the open complex formed by RNA polymerase on DNA and therefore affect promoter clearance. Its presence also leads to an increase in pausing during transcription elongation
and it competes with nucleoside triphosphate
substrate
s.
When the amino acid balance in the cell is restored, (p)ppGpp is hydrolysed
by SpoT
.
There is now a consensus that (p)ppGpp is a determinant of growth rate control rather than nucleoside triphosphate (NTP) substrate concentrations.
(p)ppGpp can affect bacterial physiology in different aspects:
A, Inhibit growth by inhibit protein synthesis. ppGpp inhibits IF2-mediated fMet-Phe initiation dipeptide formation, probably by interfering with 30S and 50S subunit interactions. E. coli accumulates more ppGpp than pppGpp during amino acid starvation, and ppGpp is about 8-fold efficiency than that of pppGpp. While B. subtilis accumulates more pppGpp than ppGpp.
B, regulating Ribosomal number.
C, inhibits DNA replication. In E. coli amino acid starvation inhibited DNA replication at the initiation stage at oriC, most probably owing to the lack of the DnaA replication initiation protein. In B. subtilis, the replication arrest due to (p)ppGpp accumulation is caused by the binding of an Rtp protein to specific sites about 100-200kb away from oriC in both directions. DNA primase (DnaG) was directly inhibited by (p)ppGpp. Unlike E. coli, B. subtilis accumulates more pppGpp than ppGpp; the more abundant nucleotide is a more-potent DnaG inhibitor. ppGpp can bind with Obg protein which belongs to the conserved, small GTPase protein family. Obg protein interacts with several regulators (RsbT, RsbW, RsbX) necessary for the stress activation of sigma B.
D, affect phage replication and development. The (p)ppGpp levels of the host seem to act as a sensor for phage lambda development, primarily affecting transcription. Modest ppGpp levels inhibit pR and active pE, pI, and paQ promoters in vivo and have effects in vitro that seem to favor lysogeny. In contrast, absent or high concentrations of (p)ppGpp favor lysis. Modest ppGpp levels favor lysogeny by leading to low HflB (FtsH). When ppGpp is either absent or high, HflB protease levels are high; this leads to lower CII
(a lysogeny-promoting phage protein)
and favors lysis.
(p)ppGpp acts at the level of transcription:
A, characteristics of affected promoters. One of the key elements of promoters inhibited by (p)ppGpp is the presence of a GC-rich discriminator, defined as a region between TATA-box (-10 box) and +1 nt (where +1 is the transcription start sit). Promoters negatively regulated by ppGpp have a 16-bp linker, in contrast with the 17-bp consensus. Promoters activated by ppGpp seem to have a AT-rich discriminator and linger linkers (for example, the his promoter linker is 18 bp).
B, RNAP is the target. Genetic evidence suggesting that RNAP was the target of ppGpp came from the discovery that M+ mutants (also called stringent RNAP mutants) display in vitro and in vivo mimicry of physiology and transcription regulation conferred by (p)ppGpp, even in its absence. Cross-linking ppGpp to RNAP reinforced this notion. Structural details of an association between ppGpp and RNAP came from the analysis of cocrystals that positioned ppGpp in the secondary channel of RNAP near the catalytic center.
C, DksA augments regulation. DksA is a 17-kDa protein, its structure is similar to GreA and GreB, which are well-characterized transcriptional elongation factors. GreA and GreB bind directly to RNAP rather than DNA and act by inserting their N-terminal coiled-coil finger domain through the RNAP secondary channel. Two conserved acidic residues at the tip of the finger domain are necessary to induce RNAP's intrinsic ability to cleave backtracked RNA. DksA also possesses two acidic residues at its finger tip, but it does not induce nucleolytic cleavage activity. Instead, these residues are proposed to stabilize ppGpp binding to RNAP by mutual coordination of an Mg2+ ion that is crucial for polymerization.
Transcription inhibition and activation:
ppGpp directly inhibits transcription from ribosomal promoters. One model is ppGpp and DksA together and independently decrease the stability of the open complexes formed on DNA by RNAP. Another model is the trapping mechanism. In this model, RNAP is trapped by ppGpp in closed complexes and is unable to initiate transcription. Thus, ppGpp seems to act at many levels, and the mechanism of its action is a complex outcome of several factors, intrinsic promoter properties not being the least of them. The the transcription activation by ppGpp can be direct or indirect. Direct activation occurs when RNAP interacts with effectors, such as ppGpp, DksA or both, to increase transcription from a given promoter. Indirect activation by these effectors of one promoter relies on inhibition of other (strong) promoters, leading to increased availability of RNAP that indirectly activates transcription initiation. The promoters that activated directly by ppGpp include PargI, PthrABC, PlivJ, and PhisG. The indirectly activation promoters include these dependent on sigma factors: S, H, N, E. When strong promoters, such as rrn, are inhibited, there more RNAP are available for these alternative sigma factors.
Pathogenesis and (p)ppGpp:
When (p)ppGpp is absent, pathogenicity is compromised fore reasons that vary with the organism studied. Deleting relA and spoT genes, but not relA alone, gave a (p)ppGpp0 state that resulted in strong attenuation in mice and noninvasiveness in vitro. Vaccine tests reveal that 30 days after single immunization with the (p)ppGpp0 strain, mice were protected from challenge with wild-type Salmonella at a dose 106-fold above the established LD50.
Alarmone
Alarmone is an intracellular signal molecule that is produced due to harsh environmental factors. They regulate the gene expression at transcription level. Alarmones are produced in high concentrations when harsh environmental factors occur in bacteria and plants, such as lack of amino acids to...
which is involved in the stringent response
Stringent response
The stringent response is a stress response that occurs in bacteria and plant chloroplasts in reaction to amino-acid starvation , fatty acid limitation , iron limitation , heat shock and other stress conditions. The stringent response is signaled by the alarmone ppGpp, and modulating...
in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, causing the inhibition of RNA synthesis when there is a shortage of amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s present. This causes translation to decrease and the amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s present are therefore conserved. Furthermore, genes for amino acid uptake (transport into the cell from surrounding media) and biosynthesis are also upregulated. However, (p)ppGpp influences the overall gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
pattern in the cell so that many genes required for survival in stressful conditions are up-regulated. A complete absence of (p)ppGpp generates its own unique phenotypic features in E. coli. These include multiple amino acid requirements, poor survival of aged cultures, aberrant cell division, morphology, and immotility, as well as being locked in a growth mode during entry into starvation.
(p)ppGpp is created via pppGpp synthase
Synthase
In biochemistry, a synthase is an enzyme that catalyses a synthesis process.Following the EC number classification, they belong to the group of ligases, with lyases catalysing the reverse reaction....
, also known as RelA, and is converted from pppGpp to ppGpp via pppGpp phosphohydrolase. RelA is associated with about every one in two hundred ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
s and it becomes activated when an uncharged transfer RNA
Transfer RNA
Transfer RNA is an adaptor molecule composed of RNA, typically 73 to 93 nucleotides in length, that is used in biology to bridge the three-letter genetic code in messenger RNA with the twenty-letter code of amino acids in proteins. The role of tRNA as an adaptor is best understood by...
(tRNA) molecule enters the A site of the ribosome, due to the shortage of amino acid required by the tRNA. If a mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
bacterium is relA- it is said to be relaxed and no regulation of RNA production due to amino acid absence is seen.
Targets of (p)ppGpp include rRNA operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
s, of which there are seven in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
(a commonly used bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
), all of which have 2 promoters. When (p)ppGpp associates with the promoter it affects the RNA polymerase
RNA polymerase
RNA polymerase is an enzyme that produces RNA. In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
's ability to bind and initiate transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
. It is thought that (p)ppGpp may affect the stability of the open complex formed by RNA polymerase on DNA and therefore affect promoter clearance. Its presence also leads to an increase in pausing during transcription elongation
Elongation
In astronomy, a planet's elongation is the angle between the Sun and the planet, as viewed from Earth. Elongations occur when an inner planet’s position, in its orbital path, is at tangent to the view from Earth. Because these inner planets are inside the Earth’s orbits their positions as viewed...
and it competes with nucleoside triphosphate
Nucleoside triphosphate
Nucleoside triphosphate is a nucleoside with three phosphates. Natural nucleoside triphosphates include adenosine triphosphate , guanosine triphosphate , cytidine triphosphate , 5-methyluridine triphosphate , and uridine triphosphate . These terms refer to those nucleoside triphosphates that...
substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
s.
When the amino acid balance in the cell is restored, (p)ppGpp is hydrolysed
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
by SpoT
SpoT
SpoT is a bacterial protein that hydrolizes alarmones, ppGpp, in the bacterial stringent response. Its hydrolase activity is Mn2+-dependent with a conserved His-Asp motif. In E coli, SpoT protein consistent of 693 amino acids. SpoT is belongs to the RSH protein family.It is bifunctional, has both...
.
There is now a consensus that (p)ppGpp is a determinant of growth rate control rather than nucleoside triphosphate (NTP) substrate concentrations.
(p)ppGpp can affect bacterial physiology in different aspects:
A, Inhibit growth by inhibit protein synthesis. ppGpp inhibits IF2-mediated fMet-Phe initiation dipeptide formation, probably by interfering with 30S and 50S subunit interactions. E. coli accumulates more ppGpp than pppGpp during amino acid starvation, and ppGpp is about 8-fold efficiency than that of pppGpp. While B. subtilis accumulates more pppGpp than ppGpp.
B, regulating Ribosomal number.
C, inhibits DNA replication. In E. coli amino acid starvation inhibited DNA replication at the initiation stage at oriC, most probably owing to the lack of the DnaA replication initiation protein. In B. subtilis, the replication arrest due to (p)ppGpp accumulation is caused by the binding of an Rtp protein to specific sites about 100-200kb away from oriC in both directions. DNA primase (DnaG) was directly inhibited by (p)ppGpp. Unlike E. coli, B. subtilis accumulates more pppGpp than ppGpp; the more abundant nucleotide is a more-potent DnaG inhibitor. ppGpp can bind with Obg protein which belongs to the conserved, small GTPase protein family. Obg protein interacts with several regulators (RsbT, RsbW, RsbX) necessary for the stress activation of sigma B.
D, affect phage replication and development. The (p)ppGpp levels of the host seem to act as a sensor for phage lambda development, primarily affecting transcription. Modest ppGpp levels inhibit pR and active pE, pI, and paQ promoters in vivo and have effects in vitro that seem to favor lysogeny. In contrast, absent or high concentrations of (p)ppGpp favor lysis. Modest ppGpp levels favor lysogeny by leading to low HflB (FtsH). When ppGpp is either absent or high, HflB protease levels are high; this leads to lower CII
CII
CII may stand for:* George M. Low Center for Industrial Innovation, a research center at Rensselaer Polytechnic Institute* The Confederation of Indian Industry* The Construction Industry Institute* Drug Schedule for the Controlled Substances Act....
(a lysogeny-promoting phage protein)
and favors lysis.
(p)ppGpp acts at the level of transcription:
A, characteristics of affected promoters. One of the key elements of promoters inhibited by (p)ppGpp is the presence of a GC-rich discriminator, defined as a region between TATA-box (-10 box) and +1 nt (where +1 is the transcription start sit). Promoters negatively regulated by ppGpp have a 16-bp linker, in contrast with the 17-bp consensus. Promoters activated by ppGpp seem to have a AT-rich discriminator and linger linkers (for example, the his promoter linker is 18 bp).
B, RNAP is the target. Genetic evidence suggesting that RNAP was the target of ppGpp came from the discovery that M+ mutants (also called stringent RNAP mutants) display in vitro and in vivo mimicry of physiology and transcription regulation conferred by (p)ppGpp, even in its absence. Cross-linking ppGpp to RNAP reinforced this notion. Structural details of an association between ppGpp and RNAP came from the analysis of cocrystals that positioned ppGpp in the secondary channel of RNAP near the catalytic center.
C, DksA augments regulation. DksA is a 17-kDa protein, its structure is similar to GreA and GreB, which are well-characterized transcriptional elongation factors. GreA and GreB bind directly to RNAP rather than DNA and act by inserting their N-terminal coiled-coil finger domain through the RNAP secondary channel. Two conserved acidic residues at the tip of the finger domain are necessary to induce RNAP's intrinsic ability to cleave backtracked RNA. DksA also possesses two acidic residues at its finger tip, but it does not induce nucleolytic cleavage activity. Instead, these residues are proposed to stabilize ppGpp binding to RNAP by mutual coordination of an Mg2+ ion that is crucial for polymerization.
Transcription inhibition and activation:
ppGpp directly inhibits transcription from ribosomal promoters. One model is ppGpp and DksA together and independently decrease the stability of the open complexes formed on DNA by RNAP. Another model is the trapping mechanism. In this model, RNAP is trapped by ppGpp in closed complexes and is unable to initiate transcription. Thus, ppGpp seems to act at many levels, and the mechanism of its action is a complex outcome of several factors, intrinsic promoter properties not being the least of them. The the transcription activation by ppGpp can be direct or indirect. Direct activation occurs when RNAP interacts with effectors, such as ppGpp, DksA or both, to increase transcription from a given promoter. Indirect activation by these effectors of one promoter relies on inhibition of other (strong) promoters, leading to increased availability of RNAP that indirectly activates transcription initiation. The promoters that activated directly by ppGpp include PargI, PthrABC, PlivJ, and PhisG. The indirectly activation promoters include these dependent on sigma factors: S, H, N, E. When strong promoters, such as rrn, are inhibited, there more RNAP are available for these alternative sigma factors.
Pathogenesis and (p)ppGpp:
When (p)ppGpp is absent, pathogenicity is compromised fore reasons that vary with the organism studied. Deleting relA and spoT genes, but not relA alone, gave a (p)ppGpp0 state that resulted in strong attenuation in mice and noninvasiveness in vitro. Vaccine tests reveal that 30 days after single immunization with the (p)ppGpp0 strain, mice were protected from challenge with wild-type Salmonella at a dose 106-fold above the established LD50.