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Escherichia coli K-12 substr. MG1655 Enzyme: GDP pyrophosphokinase / GTP pyrophosphokinase



Gene: relA Accession Numbers: EG10835 (EcoCyc), b2784, ECK2778

Synonyms: RC, stringent factor, ppGpp synthetase I, PSI, ppGpp synthase I, (p)ppGpp synthetase I

Regulation Summary Diagram: ?

Summary:
RelA is a key enzyme involved in the stringent response of Escherichia coli to amino acid starvation. It activates the synthesis of the global regulatory molecules of the stringent response ppGpp and pppGpp (referred to collectively as (p)ppGpp) via a ribosomal mechanism (see below). The amino acid sequence of RelA has been shown to be extensively related to that of SpoT [Metzger89]. The RelA (p)ppGpp synthetase activity is referred to as (p)ppGpp synthetase I, or PSI. RelA also contains an inactive hydrolase domain (reviewed in [Potrykus08]). Its catalytic activity is located in the N-terminal portion of the molecule, while the C-terminal portion may have a regulatory function [Gropp01]. The bifunctional SpoT protein provides opposing hydrolase activity for RelA synthetase activity, both of which are involved in the regulation of (p)ppGpp levels. SpoT also has a RelA-independent ppGpp synthetase activity, referred to as PSII, that can be demonstrated in relA null mutants (reviewed in Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96]).

Homologs of RelA and SpoT are members of the RelA/SpoT Homolog (RSH) superfamily. This superfamily has been divided into 30 subgroups comprising long RSHs, small alarmone synthetases (SASs) and small alarmone hydrolases (SAHs). Their phylogenetic relationships were studied across all domains of life [Atkinson11].

The mechanism of action of RelA has been studied both genetically and biochemically. It is a ribosome-associated (p)ppGpp synthetase that is activated by the binding of uncharged tRNA to its cognate codon on the acceptor site (A site) of a translating ribosome in response to amino acid starvation [Haseltine73, Payoe11]. Under these conditions ppGpp is synthesized and accumulates and stable RNA synthesis (rRNA, tRNA) is inhibited. This inhibition of stable RNA synthesis during the stringent response is "relaxed" in relA mutants (reviewed in Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96]).

Detailed in vitro studies using recombinant RelA have allowed models of RelA function to be proposed. In one study it was determined that although the presence of a deacylated tRNA on the ribosomal A site was essential for RelA-mediated ppGpp synthesis, the binding of RelA to ribosomes was dependent upon the presence of a protruding 3' extension of mRNA on the ribosome. RelA binding was independent of ribosomal protein L11, the product of gene relC (rplK). Synthesis of ppGpp by RelA reduced its binding to the ribosome. These findings led to a model in which RelA may hop between blocked ribosomal complexes during the stringent response, allowing a relatively low concentration of the enzyme to produce sufficient ppGpp [Wendrich02]. Another study implicated ribosomal protein L11 in the regulation of RelA activity [Yang01a]. Antiobiotics that block ribosome function at different sites, such as thiostrepton and tetracycline, inhibit pppGpp synthesis [Knutsson05].

More recently, an in vivo, single molecule tracking methodology was developed to monitor the intracellular RelA catalytic cycle using fluorescence imaging. Results suggested that RelA is ribosome-associated under nonstarvation conditions, but dissociates for an extended time period after activation to perform its catalytic cycle. Ribosome association was not necessary to trigger each ppGpp biosynthesis cycle. Heat stress caused fast activation of RelA with a rapid return to its inactive state, suggesting an excitable response mechanism for rapid adaptation to environmental changes [English11].

Two studies suggested that oligomerization, or dimerization of RelA via its C-terminal domain (amino acid residues 456-744) may have a role in regulation of (p)ppGpp synthetase activity. They supported a model in which oligomeric RelA is enzymatically inactive and is activated during the stringent response by dissociation to a monomer [Gropp01, Yang01c].

Apart from its known function, mutant studies have also suggested that RelA may have other as yet undefined roles in ribosome function apart from its (p)ppGpp synthetase activity [Kim09h]. It has also been shown that E. coli RelA carries a functionally important EXDD motif that has been implicated in a preference for GDP as pyrophosphate acceptor. This is in contrast to earlier reports that GTP is preferred. The EXDD motif may also provide this enzyme with the ability to synthesize pGpp at a low level, the significance of which has yet to be determined [Sajish09]. Several relA mutant alleles were shown to confer temperature-sensitive phenotypes, suggesting that ppGpp may be required for the expression of genes involved in thermotolerance [Yang03].

Native RelA has been purified to homogeneity [Pedersen77]. By itself it has little activity and must be activated in vitro by a complex of 70S ribosomes, mRNA and uncharged tRNA. In a non-ribosomal based assay it can be activated by solvents such as ethanol or methanol at concentrations of about 20% [Justesen86].

A relA spoT mutant was found to form biofilms with reduced catalase activity and elevated levels of hydroxyl radicals; the mutant biofilms are more sensitive to the antibiotics ofloxacin and tobramycin than wild type [Nguyen11].

Gene Citations: [Masuda93a]

Locations [Comment 1]: cytosol

Map Position: [2,909,439 <- 2,911,673] (62.71 centisomes)
Length: 2235 bp / 744 aa

Molecular Weight of Polypeptide: 83.876 kD (from nucleotide sequence), 84.0 kD (experimental) [Metzger88 ]

pI: 6.76

Isozyme Sequence Similarity:
guanosine 3'-diphosphate 5'-triphosphate 3'-diphosphatase [multifunctional]: YES

Unification Links: ASAP:ABE-0009125 , CGSC:306 , EchoBASE:EB0828 , EcoGene:EG10835 , EcoliWiki:b2784 , ModBase:P0AG20 , OU-Microarray:b2784 , PortEco:relA , PR:PRO_000023710 , Pride:P0AG20 , Protein Model Portal:P0AG20 , RefSeq:NP_417264 , RegulonDB:EG10835 , SMR:P0AG20 , String:511145.b2784 , UniProt:P0AG20

Relationship Links: InterPro:IN-FAMILY:IPR002912 , InterPro:IN-FAMILY:IPR004095 , InterPro:IN-FAMILY:IPR004811 , InterPro:IN-FAMILY:IPR007685 , InterPro:IN-FAMILY:IPR012675 , InterPro:IN-FAMILY:IPR012676 , InterPro:IN-FAMILY:IPR026020 , Panther:IN-FAMILY:PTHR21262 , Panther:IN-FAMILY:PTHR21262:SF1 , Pfam:IN-FAMILY:PF01842 , Pfam:IN-FAMILY:PF02824 , Pfam:IN-FAMILY:PF04607 , Prosite:IN-FAMILY:PS51671 , Smart:IN-FAMILY:SM00954

In Paralogous Gene Group: 457 (2 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0015949 - nucleobase-containing small molecule interconversion Inferred from experiment [Wendrich02]
GO:0015969 - guanosine tetraphosphate metabolic process Inferred from experiment Inferred by computational analysis [GOA01, Metzger89a]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01]
GO:0015970 - guanosine tetraphosphate biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0008728 - GTP diphosphokinase activity Inferred from experiment Inferred by computational analysis [GOA01a, Pedersen77]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0005525 - GTP binding Inferred by computational analysis [UniProtGOA11]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11]
GO:0016597 - amino acid binding Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment [Pedersen77]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: cell structure ribosomes
metabolism central intermediary metabolism nucleotide and nucleoside conversions
regulation type of regulation unknown

Essentiality data for relA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 2]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 3]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 4]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 3]
Yes [Feist07, Comment 5]

Credits:
Last-Curated ? 22-Nov-2011 by Fulcher C , SRI International


Enzymatic reaction of: GDP pyrophosphokinase

Synonyms: ppGpp synthase, ppGpp synthetase

EC Number: 2.7.6.5

ATP + GDP <=> AMP + ppGpp

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown. [Justesen86]

In Pathways: ppGpp biosynthesis

Summary:
In this reaction the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) involves transfer of the β,γ-phosphates (the terminal pyrophosphoryl group) from ATP to the ribose 3'-hydroxyl of GDP. Although the reaction mechanism is complex and ribosome-dependent, the synthetic activity of purified RelA can be activated in vitro by methanol in the absence of ribosomal factors. These two observations were first demonstrated in Escherichia coli K-19 [Sy73, Sy73a]. Reviewed in Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96].

Inhibitors (Unknown Mechanism): ppGpp [Comment 6]


Enzymatic reaction of: GTP pyrophosphokinase

Synonyms: guanosine 3',5'-polyphosphate synthase, ATP:GTP 3'-pyrophosphotransferase, GTP diphosphokinase, ATP:GTP 3'-diphosphotransferase, guanosine 3',5'-polyphosphate synthetase, guanosine pentaphosphate synthetase

EC Number: 2.7.6.5

GTP + ATP <=> pppGpp + AMP

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown. [Justesen86]

In Pathways: ppGpp biosynthesis

Summary:
In this reaction the synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) involves transfer of the β,γ-phosphates (the terminal pyrophosphoryl group) from ATP to the ribose 3'-hydroxyl of GTP. Although the reaction mechanism is complex and ribosome-dependent, the synthetic activity of purified RelA can be activated in vitro by methanol in the absence of ribosomal factors, as first demonstrated in Escherichia coli K-19 [Sy73a]. Reviewed in Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96].

Although physiologically favored in the synthetic direction, this reaction was shown to be reversible in vitro using unpurified enzyme from Escherichia coli K-19. The reverse reaction preferentially used pppGpp rather than ppGpp [Sy74].


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 307
[Metzger88, UniProt10]
Alternate sequence: F → K; UniProt: (in Ref. 1; AAA03237);
Conserved-Region 668 -> 743
[UniProt13]
UniProt: ACT.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b2784 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10835; confirmed by SwissProt match.


References

Atkinson11: Atkinson GC, Tenson T, Hauryliuk V (2011). "The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life." PLoS One 6(8);e23479. PMID: 21858139

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

English11: English BP, Hauryliuk V, Sanamrad A, Tankov S, Dekker NH, Elf J (2011). "Single-molecule investigations of the stringent response machinery in living bacterial cells." Proc Natl Acad Sci U S A 108(31);E365-73. PMID: 21730169

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

Gropp01: Gropp M, Strausz Y, Gross M, Glaser G (2001). "Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain." J Bacteriol 183(2);570-9. PMID: 11133950

Haseltine73: Haseltine WA, Block R (1973). "Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes." Proc Natl Acad Sci U S A 70(5);1564-8. PMID: 4576025

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Justesen86: Justesen J, Lund T, Skou Pedersen F, Kjeldgaard NO (1986). "The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli." Biochimie 1986;68(5);715-22. PMID: 3015258

Kim09h: Kim HM, Ryou SM, Song WS, Sim SH, Cha CJ, Han SH, Ha NC, Kim JH, Bae J, Cunningham PR, Lee K (2009). "Genetic analysis of the invariant residue G791 in Escherichia coli 16S rRNA implicates RelA in ribosome function." J Bacteriol 191(7);2042-50. PMID: 19168615

Knutsson05: Knutsson Jenvert RM, Holmberg Schiavone L (2005). "Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli." FEBS J 272(3);685-95. PMID: 15670150

Masuda93a: Masuda Y, Miyakawa K, Nishimura Y, Ohtsubo E (1993). "chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100." J Bacteriol 1993;175(21);6850-6. PMID: 8226627

Metzger88: Metzger S, Dror IB, Aizenman E, Schreiber G, Toone M, Friesen JD, Cashel M, Glaser G (1988). "The nucleotide sequence and characterization of the relA gene of Escherichia coli." J Biol Chem 1988;263(30);15699-704. PMID: 2844820

Metzger89: Metzger S, Sarubbi E, Glaser G, Cashel M (1989). "Protein sequences encoded by the relA and the spoT genes of Escherichia coli are interrelated." J Biol Chem 1989;264(16);9122-5. PMID: 2542299

Metzger89a: Metzger S, Schreiber G, Aizenman E, Cashel M, Glaser G (1989). "Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli." J Biol Chem 264(35);21146-52. PMID: 2556396

Neidhardt96: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low Jr KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE "Escherichia coli and Salmonella, Cellular and Molecular Biology, Second Edition." American Society for Microbiology, Washington, D.C., 1996.

Nguyen11: Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, McKay G, Siehnel R, Schafhauser J, Wang Y, Britigan BE, Singh PK (2011). "Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria." Science 334(6058);982-6. PMID: 22096200

Payoe11: Payoe R, Fahlman RP (2011). "Dependence of RelA-mediated (p)ppGpp formation on tRNA identity." Biochemistry 50(15);3075-83. PMID: 21410133

Pedersen77: Pedersen FS, Kjeldgaard NO (1977). "Analysis of the relA gene product of Escherichia coli." Eur J Biochem 76(1);91-7. PMID: 195816

Potrykus08: Potrykus K, Cashel M (2008). "(p)ppGpp: still magical?." Annu Rev Microbiol 62;35-51. PMID: 18454629

Sajish09: Sajish M, Kalayil S, Verma SK, Nandicoori VK, Prakash B (2009). "The significance of EXDD and RXKD motif conservation in Rel proteins." J Biol Chem 284(14);9115-23. PMID: 19201753

Sy73: Sy J, Lipmann F (1973). "Identification of the synthesis of guanosine tetraphosphate (MS I) as insertion of a pyrophosphoryl group into the 3'-position in guanosine 5'-diphosphate." Proc Natl Acad Sci U S A 70(2);306-9. PMID: 4346881

Sy73a: Sy J, Ogawa Y, Lipmann F (1973). "Nonribosomal synthesis of guanosine 5',3'-polyphosphates by the ribosomal wash of stringent Escherichia coli." Proc Natl Acad Sci U S A 70(7);2145-8. PMID: 4579015

Sy74: Sy J (1974). "Reversibility of the pyrophosphoryl transfer from ATP to GTP by Escherichia coli stringent factor." Proc Natl Acad Sci U S A 71(9);3470-3. PMID: 4372621

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Wendrich02: Wendrich TM, Blaha G, Wilson DN, Marahiel MA, Nierhaus KH (2002). "Dissection of the mechanism for the stringent factor RelA." Mol Cell 10(4);779-88. PMID: 12419222

Yang01a: Yang X, Ishiguro EE (2001). "Involvement of the N terminus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli." J Bacteriol 183(22);6532-7. PMID: 11673421

Yang01c: Yang X, Ishiguro EE (2001). "Dimerization of the RelA protein of Escherichia coli." Biochem Cell Biol 79(6);729-36. PMID: 11800013

Yang03: Yang X, Ishiguro EE (2003). "Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli." J Bacteriol 185(19);5765-71. PMID: 13129947

Other References Related to Gene Regulation

Goodman12: Goodman C (2012). "Regulation: positively alarming." Nat Chem Biol 8(9);738. PMID: 22907080

Lin13: Lin CY, Awano N, Masuda H, Park JH, Inouye M (2013). "Transcriptional Repressor HipB Regulates the Multiple Promoters in Escherichia coli." J Mol Microbiol Biotechnol 23(6);440-447. PMID: 24089053

Maciag11: Maciag A, Peano C, Pietrelli A, Egli T, De Bellis G, Landini P (2011). "In vitro transcription profiling of the {sigma}S subunit of bacterial RNA polymerase: re-definition of the {sigma}S regulon and identification of {sigma}S-specific promoter sequence elements." Nucleic Acids Res 39(13);5338-55. PMID: 21398637

Montero09: Montero M, Eydallin G, Viale AM, Almagro G, Munoz FJ, Rahimpour M, Sesma MT, Baroja-Fernandez E, Pozueta-Romero J (2009). "Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes." Biochem J 424(1);129-41. PMID: 19702577

Nakagawa06: Nakagawa A, Oshima T, Mori H (2006). "Identification and characterization of a second, inducible promoter of relA in Escherichia coli." Genes Genet Syst 81(5);299-310. PMID: 17159291

Traxler08: Traxler MF, Summers SM, Nguyen HT, Zacharia VM, Hightower GA, Smith JT, Conway T (2008). "The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli." Mol Microbiol 68(5);1128-48. PMID: 18430135


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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