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Escherichia coli K-12 substr. MG1655 Enzyme: tryptophanyl-tRNA synthetase



Gene: trpS Accession Numbers: EG11030 (EcoCyc), b3384, ECK3371

Synonyms: TrpRS

Regulation Summary Diagram: ?

Subunit composition of tryptophanyl-tRNA synthetase = [TrpS]2
         tryptophanyl-tRNA synthetase = TrpS

Summary:
Tryptophanyl-tRNA synthetase (TrpRS) is a member of the family of aminoacyl-tRNA synthetases, which interpret the genetic code by covalently linking amino acids to their specific tRNA molecules. The reaction is driven by ATP hydrolysis. TrpRS belongs to the Class I aminoacyl tRNA synthetases [Eriani90, Landes95].

The enzyme purified from E. coli B is a homodimer in solution [Joseph71, Joseph71a]. The dimer has two binding sites for both tryptophan and tRNATrp [Muench76].

Specificity determinants within tRNATrp that are important for recognition by TrpRS have been identified. The anticodon and the G73 discriminator base are the major identity determinants [Himeno91], and C35 is recognized as well [Rogers92]. The apparent affinity of TrpRS for tryptophan appears to be dependent on the tRNA [Ibba96]. The identity of tRNATrp predominantly affects the rate of transfer of tryptophan from the TrpRS-tryptophanyl adenylate to the tRNA [Ibba99]. TrpRS also aminoacylates tRNATrp with D-tryptophan; the resulting D-Trp-tRNATrp can be hydrolyzed by D-Tyr-tRNATyr deacylase [Soutourina00]. A strain that can incorporate 4-fluorotryptophan in place of tryptophan into proteins has been isolated and contains, among others, mutations in TrpRS [Bacher01].

The sequential processes involved in the tRNA charging reaction have been studied [Andrews85, Merle86, Lloyd95]. Mutagenesis of the Thr17 residue in the TIGN motif indicates that Thr17 is important for binding of substrate in the transition state [Chan94], and mutagenesis of Lys195 in the conserved KMSKS motif indicates that Lys195 may interact with ATP in the transition state [Chan95a]. Mutants that are auxotrophic for tryptophan and map within trpS have been identified; the mutations are located within the conserved KMSKS motif, near the active site, or lining a proposed dimerization interface, supporting a role for dimerization of the enzyme in catalysis [Sever96].

TrpRS activity increases with the growth rate; the regulation is at the level of trpS transcription [Hall82a].

Reviews: [Ibba00, Giege12, Perona12]

Citations: [Ito69b, Ito69c, Ito72, Squires73, Morse76, Kuehl76, Winter77, Bohman78, Drocourt79, Hall82b, Bogosian83, Das84, Koeppe85, Yamao88, Yamao88a, Popenko92, Azim08]

Gene Citations: [Lyngstadaas95, Lyngstadaas99]

Locations: cytosol

Map Position: [3,510,656 <- 3,511,660] (75.67 centisomes)
Length: 1005 bp / 334 aa

Molecular Weight of Polypeptide: 37.438 kD (from nucleotide sequence), 37 kD (experimental) [Hall81 ]

Molecular Weight of Multimer: 74 kD (experimental) [Joseph71]

pI: 6.2 [Joseph71]

Unification Links: ASAP:ABE-0011056 , CGSC:67 , DIP:DIP-11042N , EchoBASE:EB1023 , EcoGene:EG11030 , EcoliWiki:b3384 , Mint:MINT-1244806 , ModBase:P00954 , OU-Microarray:b3384 , PortEco:trpS , PR:PRO_000024125 , Pride:P00954 , Protein Model Portal:P00954 , RefSeq:NP_417843 , RegulonDB:EG11030 , SMR:P00954 , String:511145.b3384 , Swiss-Model:P00954 , UniProt:P00954

Relationship Links: InterPro:IN-FAMILY:IPR001412 , InterPro:IN-FAMILY:IPR002305 , InterPro:IN-FAMILY:IPR002306 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR024109 , Panther:IN-FAMILY:PTHR10055 , Pfam:IN-FAMILY:PF00579 , Prints:IN-FAMILY:PR01039 , Prosite:IN-FAMILY:PS00178

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006436 - tryptophanyl-tRNA aminoacylation Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Doolittle68]
GO:0006412 - translation Inferred by computational analysis [UniProtGOA11]
GO:0006418 - tRNA aminoacylation for protein translation Inferred by computational analysis [GOA01a]
Molecular Function: GO:0004830 - tryptophan-tRNA ligase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Penzer74]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0004812 - aminoacyl-tRNA ligase activity Inferred by computational analysis [UniProtGOA11, GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11, GOA06, GOA01a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Popenko93]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06, GOA01a]

MultiFun Terms: information transfer protein related amino acid -activation

Essentiality data for trpS knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Credits:
Last-Curated ? 23-Apr-2014 by Keseler I , SRI International


Enzymatic reaction of: tryptophanyl-tRNA synthetase

Synonyms: tryptophan--tRNA ligase

EC Number: 6.1.1.2

L-tryptophan + tRNAtrp + ATP + H+ <=> L-tryptophanyl-tRNAtrp + AMP + diphosphate

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.

Alternative Substrates for L-tryptophan: D-tryptophan [Soutourina00 ]

In Pathways: tRNA charging

Summary:
Some properties of TrpRS were first measured using enzyme purified from E. coli B [Joseph71].

Citations: [Andrews85, Merle86, Lloyd95]

Cofactors or Prosthetic Groups: Mg2+ [Penzer74]

Inhibitors (Competitive): tryptophanhydroxamate [Gao95] , indolmycin (Kic = 8.5µM) [Werner76]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
L-tryptophan
17.0
[Zuniga02, BRENDA14]
L-tryptophan
12.4
2.0
[Sever96]
ATP
200.0
[Drocourt79, BRENDA14]
ATP
190.0
1.5
[Sever96]
tRNAtrp
0.34
2.0
[Sever96]

pH(opt): 8.8 [Joseph71]


Sequence Features

Feature Class Location Citations Comment
Protein-Segment 12 -> 20
[UniProt10a]
UniProt: "HIGH" region; Sequence Annotation Type: short sequence motif;
Sequence-Conflict 30
[Hall82b, Lyngstadaas95, UniProt10a]
Alternate sequence: N → K; UniProt: (in Ref. 1 and 2);
Extrinsic-Sequence-Variant 60
[UniProt10a]
Alternate sequence: T → R; UniProt: (in TRPS567C; reduced activity, thermolabile);
Extrinsic-Sequence-Variant 91
[UniProt10a]
Alternate sequence: L → F; UniProt: (in TRPS10330; reduced activity);
Extrinsic-Sequence-Variant 112
[UniProt10a]
Alternate sequence: D → E; UniProt: (in TRPS9969; no effect in activity);
Extrinsic-Sequence-Variant 129
[UniProt10a]
Alternate sequence: P → S; UniProt: (in TRPS9969; reduced activity);
Extrinsic-Sequence-Variant 133
[UniProt10a]
Alternate sequence: A → E; UniProt: (in TRPS42C; reduced activity);
Protein-Segment 195 -> 199
[UniProt10a]
UniProt: "KMSKS" region; Sequence Annotation Type: short sequence motif;
Extrinsic-Sequence-Variant 196
[UniProt10a]
Alternate sequence: M → I; UniProt: (in TRPS271C; activity largely reduced);
Amino-Acid-Sites-That-Bind 198
[UniProt10]
UniProt: ATP; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 326
[Hall82b, Lyngstadaas95, UniProt10a]
Alternate sequence: E → Q; UniProt: (in Ref. 1 and 2);
Extrinsic-Sequence-Variant 329
[UniProt10a]
Alternate sequence: G → S; UniProt: (in TRPS4040; reduced activity);
Sequence-Conflict 334
[Sever96, Hall82b, Lyngstadaas95, UniProt10a]
Alternate sequence: P → R; UniProt: (in Ref. 1, 2 and 3);


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b3384 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11030; confirmed by SwissProt match.


References

Andrews85: Andrews D, Trezeguet V, Merle M, Graves PV, Muench KH, Labouesse B (1985). "Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase." Eur J Biochem 146(1);201-9. PMID: 3881255

Azim08: Azim MK, Budisa N (2008). "Docking of tryptophanyl [corrected tryptophan] analogs to trytophanyl-tRNA synthetase: implications for non-canonical amino acid incorporations." Biol Chem 389(9);1173-82. PMID: 18713004

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

Bacher01: Bacher JM, Ellington AD (2001). "Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue." J Bacteriol 183(18);5414-25. PMID: 11514527

Bogosian83: Bogosian G, Haydock PV, Somerville RL (1983). "Indolmycin-mediated inhibition and stimulation of transcription at the trp promoter of Escherichia coli." J Bacteriol 153(2);1120-3. PMID: 6337120

Bohman78: Bohman K, Isaksson LA (1978). "Mutations in the tryptophanyl-transfer ribonucleic acid ligase of E. coli causing temperature-sensitivity for growth." Mol Gen Genet 161(3);285-9. PMID: 353515

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Chan94: Chan KW, Koeppe RE (1994). "Role of the TIGN sequence in E. coli tryptophanyl-tRNA synthetase." Biochim Biophys Acta 1205(2);223-9. PMID: 8155701

Chan95a: Chan KW, Koeppe RE (1995). "Role of lysine-195 in the KMSKS sequence of E. coli tryptophanyl-tRNA synthetase." FEBS Lett 363(1-2);33-6. PMID: 7729548

Das84: Das A, Yanofsky C (1984). "Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator." J Bacteriol 160(2);805-7. PMID: 6389500

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

Doolittle68: Doolittle WF, Yanofsky C (1968). "Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase." J Bacteriol 95(4);1283-94. PMID: 4869215

Drocourt79: Drocourt JL, Thang DC, Buckingham RH, Thang MN (1979). "Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme." Nucleic Acids Res 6(8);2919-28. PMID: 379831

Eriani90: Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990). "Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs." Nature 347(6289);203-6. PMID: 2203971

Gao95: Gao W, Jakubowski H, Goldman E (1995). "Evidence that uncharged tRNA can inhibit a programmed translational frameshift in Escherichia coli." J Mol Biol 251(2);210-6. PMID: 7643397

Giege12: Giege R, Springer M (2012). "Aminoacyl-tRNA Synthetases in the Bacterial World." EcoSal online, chapter 4.2.1.

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Hall81: Hall CV, Yanofsky C (1981). "Cloning and characterization of the gene for Escherichia coli tryptophanyl-transfer ribonucleic acid synthetase." J Bacteriol 148(3);941-9. PMID: 6171561

Hall82a: Hall CV, Yanofsky C (1982). "Regulation of tryptophanyl-tRNA synthetase formation." J Bacteriol 151(2);918-23. PMID: 7047500

Hall82b: Hall CV, vanCleemput M, Muench KH, Yanofsky C (1982). "The nucleotide sequence of the structural gene for Escherichia coli tryptophanyl-tRNA synthetase." J Biol Chem 257(11);6132-6. PMID: 7042706

Himeno91: Himeno H, Hasegawa T, Asahara H, Tamura K, Shimizu M (1991). "Identity determinants of E. coli tryptophan tRNA." Nucleic Acids Res 19(23);6379-82. PMID: 1721699

Ibba00: Ibba M, Soll D (2000). "Aminoacyl-tRNA synthesis." Annu Rev Biochem 69;617-50. PMID: 10966471

Ibba96: Ibba M, Hong KW, Sherman JM, Sever S, Soll D (1996). "Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme." Proc Natl Acad Sci U S A 93(14);6953-8. PMID: 8692925

Ibba99: Ibba M, Sever S, Praetorius-Ibba M, Soll D (1999). "Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis." Nucleic Acids Res 27(18);3631-7. PMID: 10471730

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Ito69b: Ito K, Hiraga S, Yura T (1969). "Tryptophanyl transfer RNA synthetase and expression of the tryptophan operon in the trpS mutants of Escherichia coli." Genetics 61(3);521-38. PMID: 4914847

Ito69c: Ito K, Hiraga S, Yura T (1969). "Temperature-sensitive repression of the tryptophan operon in Escherichia coli." J Bacteriol 99(1);279-86. PMID: 4895848

Ito72: Ito K (1972). "Regulatory mechanism of the tryptophan operon in Escherichia coli: possible interaction between trpR and trpS gene products." Mol Gen Genet 115(4);349-63. PMID: 4555910

Joseph71: Joseph DR, Muench KH (1971). "Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. I. Purification of the enzyme and of tryptrophan transfer ribonucleic acid." J Biol Chem 246(24);7602-9. PMID: 4332555

Joseph71a: Joseph DR, Muench KH (1971). "Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. II. Molecular weight, subunit structure, sulfhydryl content, and substrate-binding properties." J Biol Chem 246(24);7610-5. PMID: 4944315

Koeppe85: Koeppe RE, Haw JH, Paczkowski JA (1985). "On the recovery of Cys-containing peptides during peptide mapping by HPLC. Tryptic peptides of Trp-tRNA synthetase of E.coli." FEBS Lett 183(2);313-6. PMID: 3886424

Kuehl76: Kuehl GV, Lee ML, Muench KH (1976). "Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides." J Biol Chem 251(11);3254-60. PMID: 776964

Landes95: Landes C, Perona JJ, Brunie S, Rould MA, Zelwer C, Steitz TA, Risler JL (1995). "A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases." Biochimie 77(3);194-203. PMID: 7647112

Lloyd95: Lloyd AJ, Thomann HU, Ibba M, Soll D (1995). "A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity." Nucleic Acids Res 23(15);2886-92. PMID: 7659511

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Lyngstadaas95: Lyngstadaas A, Lobner-Olesen A, Boye E (1995). "Characterization of three genes in the dam-containing operon of Escherichia coli." Mol Gen Genet 1995;247(5);546-54. PMID: 7603433

Lyngstadaas99: Lyngstadaas A, Lobner-Olesen A, Grelland E, Boye E (1999). "The gene for 2-phosphoglycolate phosphatase (gph) in Escherichia coli is located in the same operon as dam and at least five other diverse genes." Biochim Biophys Acta 1472(1-2);376-84. PMID: 10572959

Merle86: Merle M, Trezeguet V, Graves PV, Andrews D, Muench KH, Labouesse B (1986). "Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli." Biochemistry 25(5);1115-23. PMID: 3516215

Morse76: Morse DE, Morse AN (1976). "Dual-control of the tryptophan operon is mediated by both tryptophanyl-tRNA synthetase and the repressor." J Mol Biol 103(2);209-26. PMID: 781267

Muench76: Muench KH (1976). "Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli." J Biol Chem 251(17);5195-9. PMID: 783157

Penzer74: Penzer GR, Plumbridge JA (1974). "Kinetics of pyrophosphate-ATP exchange catalysed by L-tryptophan: tRNA ligase from Escherichia coli." Eur J Biochem 45(1);291-5. PMID: 4371652

Perona12: Perona JJ, Hadd A (2012). "Structural diversity and protein engineering of the aminoacyl-tRNA synthetases." Biochemistry 51(44);8705-29. PMID: 23075299

Popenko92: Popenko VI, Cherin NE, Ivanova IuL, Beresten' SF, Filonenko VV (1992). "[Immunoelectron-microscopic determination of the localization of tryptophanyl-tRNA synthetase in eubacterial cells of Escherichia coli and Methanococcus halophilus archaebacteria]." Mol Biol (Mosk) 26(1);83-92. PMID: 1508173

Popenko93: Popenko VI, Cherny NE, Beresten SF, Ivanova JL, Filonenko VV, Kisselev LL (1993). "Immunoelectron microscopic location of tryptophanyl-tRNA synthetase in mammalian, prokaryotic and archaebacterial cells." Eur J Cell Biol 62(2);248-58. PMID: 7925483

Rogers92: Rogers MJ, Adachi T, Inokuchi H, Soll D (1992). "Switching tRNA(Gln) identity from glutamine to tryptophan." Proc Natl Acad Sci U S A 89(8);3463-7. PMID: 1565639

Sever96: Sever S, Rogers K, Rogers MJ, Carter C, Soll D (1996). "Escherichia coli tryptophanyl-tRNA synthetase mutants selected for tryptophan auxotrophy implicate the dimer interface in optimizing amino acid binding." Biochemistry 35(1);32-40. PMID: 8555191

Soutourina00: Soutourina J, Plateau P, Blanquet S (2000). "Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells." J Biol Chem 275(42);32535-42. PMID: 10918062

Squires73: Squires CL, Rose JK, Yanofsky C, Yang HL, Zubay G (1973). "Tryptophanyl-tRNA and tryptophanyl-tRNA synthetase are not required for in vitro repression of the tryptophan operon." Nat New Biol 245(144);131-3. PMID: 4582891

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

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

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

Werner76: Werner RG, Thorpe LF, Reuter W, Nierhaus KH (1976). "Indolmycin inhibits prokaryotic tryptophanyl-tRNA ligase." Eur J Biochem 68(1);1-3. PMID: 786633

Winter77: Winter GP, Hartley BS, McLachlan AD, Lee M, Muench KH (1977). "Sequence homologies between the tryptophanyl tRNA synthetases of Bacillus stearothermophilus and Escherichia coli." FEBS Lett 82(2);348-50. PMID: 334569

Yamao88: Yamao F, Inokuchi H, Ozeki H (1988). "Mischarging mutants of Su+2 glutamine tRNA in E. coli. I. Mutations near the anticodon cause mischarging." Jpn J Genet 63(3);237-49. PMID: 3078873

Yamao88a: Yamao F, Inokuchi H, Normanly J, Abelson J, Ozeki H (1988). "Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs." Jpn J Genet 63(3);251-8. PMID: 3078874

Zuniga02: Zuniga R, Salazar J, Canales M, Orellana O (2002). "A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding." FEBS Lett 532(3);387-90. PMID: 12482597

Other References Related to Gene Regulation

LobnerOlesen92a: Lobner-Olesen A, Boye E, Marinus MG (1992). "Expression of the Escherichia coli dam gene." Mol Microbiol 1992;6(13);1841-51. PMID: 1630320

Wu92c: Wu TH, Grelland E, Boye E, Marinus MG (1992). "Identification of a weak promoter for the dam gene of Escherichia coli." Biochim Biophys Acta 1992;1131(1);47-52. PMID: 1581360


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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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