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Escherichia coli K-12 substr. MG1655 Enzyme: tRNA-specific 2-thiouridylase



Gene: mnmA Accession Numbers: EG11344 (EcoCyc), b1133, ECK1119

Synonyms: asuE, ycfB, trmU

Regulation Summary Diagram: ?

Summary:
MnmA catalyzes formation of the 2-thiouridine modification of the modified nucleoside 5-methylamino-methyl-2-thiouridine (mnm5s2U34) in the wobble position of tRNAGln, tRNALys and tRNAGlu [Kambampati03, Ikeuchi06].

A sulfur relay system consisting of IscS and the TusABCDE proteins is required for delivery of the sulfur atom [Ikeuchi06]. In vitro, a low level of MnmA activity on tRNAGlu could be achieved in the presence of Mg-ATP, cysteine, and the IscS cysteine desulfurase. The enzyme also shows activity toward anticodon stem-loop (tRNA fragment) substrates. MnmA binds to tRNALys and appears to bind to ATP [Kambampati03].

Crystal structures of MnmA together with tRNAGlu have been solved [Numata06a, Numata06b], revealing an adenylated tRNA intermediate and the basis for recognition of the specific tRNA substrates. Possible reaction mechanisms were proposed [Numata06b].

One published report claims that mnmA is essential [Arigoni98], while later studies report that it is not [Dassain99, Nilsson02, Kambampati03]. mnmA mutants show a slow growth phenotype [Dassain99, Nilsson02, Kambampati03].

An mnmA mutation decreases supL nonsense suppression by causing a defect in 5-methylaminomethyl-2-thiouridine modification (probably a defect in 2-thiolation) of the suppressor tRNALys [Sullivan85a]. An mnmA mutant exhibits increased frameshifting, compared to wild type, indicating that modified nucleosides in tRNAs act to ensure consistent translation in the proper reading frame [Urbonavicius01], but has only a small effect on ribosomal frameshifting at an exogenous frameshifting "slippery" sequence [Brierley97]. In contrast to +1 frameshifting, a mnmA mutation has no effect on -1 frameshifting [Urbonavicius03]. Contrary to expectations, an mnmA mutant shows decreased mis-recognition by tRNALys of Asp codons, and does not appear to cause a defect in lysine charging of the tRNALys in vivo [Hagervall98].

AsuE: "antisuppressor E" [Sullivan85a]

Reviews: [Rogers95a, Li07b]

Locations: cytosol

Map Position: [1,191,890 <- 1,192,996] (25.69 centisomes)
Length: 1107 bp / 368 aa

Molecular Weight of Polypeptide: 40.959 kD (from nucleotide sequence), 45.0 kD (experimental) [Kambampati03 ]

Unification Links: ASAP:ABE-0003815 , CGSC:37613 , DIP:DIP-11035N , EchoBASE:EB1320 , EcoGene:EG11344 , EcoliWiki:b1133 , ModBase:P25745 , OU-Microarray:b1133 , PortEco:mnmA , PR:PRO_000023254 , Pride:P25745 , Protein Model Portal:P25745 , RefSeq:NP_415651 , RegulonDB:EG11344 , SMR:P25745 , String:511145.b1133 , UniProt:P25745

Relationship Links: InterPro:IN-FAMILY:IPR004506 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR023382 , Panther:IN-FAMILY:PTHR11933 , PDB:Structure:2DER , PDB:Structure:2DET , PDB:Structure:2DEU , Pfam:IN-FAMILY:PF03054

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0002143 - tRNA wobble position uridine thiolation Inferred from experiment [Kambampati03, Sullivan85a]
GO:0006400 - tRNA modification Inferred by computational analysis [GOA06]
GO:0008033 - tRNA processing Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0016783 - sulfurtransferase activity Inferred from experiment Inferred by computational analysis [GOA01a, Kambampati03]
GO:0000049 - tRNA binding Inferred by computational analysis [UniProtGOA11a]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003723 - RNA binding Inferred by computational analysis [UniProtGOA11a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, GOA01a]

MultiFun Terms: information transfer RNA related RNA modification

Essentiality data for mnmA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba et al., 2006, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba et al., 2006, Comment 1]

Credits:
Last-Curated ? 22-Sep-2011 by Keseler I , SRI International


Enzymatic reaction of: tRNA-specific 2-thiouridylase

EC Number: 6.-.-.-

a tRNA uridine34 + a [TusE sulfur carrier protein]-S-sulfanylcysteine + ATP + H+ <=> a tRNA 2-thiouridine34 + a [TusE sulfur carrier protein]-L-cysteine + AMP + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Summary:
The IscS cysteine desulfurase [Kambampati03] and the Tus proteins [Ikeuchi06] are required for this reaction.


Sequence Features

Feature Class Location Citations Comment
Nucleotide-Phosphate-Binding-Region 11 -> 18
[UniProt10]
UniProt: ATP;
Mutagenesis-Variant 17
[Numata06b, UniProt11a]
Alternate sequence: D → A; UniProt: Loss of activity.
Mutagenesis-Variant 37
[Numata06b, UniProt11a]
Alternate sequence: M → A; UniProt: Reduces activity by 60%.
Amino-Acid-Sites-That-Bind 37
[UniProt10]
UniProt: ATP; via amide nitrogen and carbonyl oxygen;
Mutagenesis-Variant 97
[Numata06b, UniProt11a]
Alternate sequence: N → A; UniProt: Loss of activity.
Protein-Segment 97 -> 99
[UniProt, 2010]
UniProt: Interaction with target base in tRNA; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 99
[Numata06b, UniProt11a]
Alternate sequence: D → A; UniProt: Loss of activity.
Mutagenesis-Variant 102
[Numata06b, UniProt11a]
Alternate sequence: C → A; UniProt: Loss of activity.
Disulfide-Bond-Site 199, 102
[UniProt10]
UniProt: Alternate;
Active-Site 102
[UniProt10]
UniProt: Nucleophile;
Mutagenesis-Variant 107
[Numata06b, UniProt11a]
Alternate sequence: K → M; UniProt: Reduces activity by 75%.
Amino-Acid-Sites-That-Bind 127
[UniProt10]
UniProt: ATP; via amide nitrogen;
Mutagenesis-Variant 128
[Numata06b, UniProt11a]
Alternate sequence: H → A; UniProt: Reduces activity by 90%.
Amino-Acid-Site 128
[UniProt10]
UniProt: Interaction with tRNA; Sequence Annotation Type: site;
Mutagenesis-Variant 149
[Numata06b, UniProt11a]
Alternate sequence: K → A; UniProt: Loss of activity.
Protein-Segment 149 -> 151
[UniProt, 2010]
UniProt: Interaction with tRNA; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 151
[Numata06b, UniProt11a]
Alternate sequence: Q → E; UniProt: Loss of activity.
Mutagenesis-Variant 199
[Numata06b, UniProt11a]
Alternate sequence: C → A; UniProt: Abolishes the incorporation of sulfur from the sulfur-relay system; loss of activity.
Active-Site 199
[UniProt10]
UniProt: Cysteine persulfide intermediate;
Mutagenesis-Variant 200
[Numata06b, UniProt11a]
Alternate sequence: F → A; UniProt: Reduces activity by 60%.
Mutagenesis-Variant 239
[Numata06b, UniProt11a]
Alternate sequence: T → A; UniProt: Reduces activity by 50%.
Protein-Segment 243 -> 252
[UniProt, 2010]
UniProt: Interaction with tRNA; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 311
[Numata06b, UniProt11a]
Alternate sequence: R → A; UniProt: Reduces activity by 75%.
Protein-Segment 311 -> 312
[UniProt, 2010]
UniProt: Interaction with tRNA; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 344
[Numata06b, UniProt11a]
Alternate sequence: Q → A; UniProt: Loss of activity.
Amino-Acid-Site 344
[UniProt10]
UniProt: Interaction with tRNA; Sequence Annotation Type: site;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
Peter D. Karp on Wed Jan 18, 2006:
Gene right-end position adjusted based on analysis performed in the 2005 E. coli annotation update [Riley06 ].
10/20/97 Gene b1133 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11344; confirmed by SwissProt match.


References

Arigoni98: Arigoni F, Talabot F, Peitsch M, Edgerton MD, Meldrum E, Allet E, Fish R, Jamotte T, Curchod ML, Loferer H (1998). "A genome-based approach for the identification of essential bacterial genes." Nat Biotechnol 16(9);851-6. PMID: 9743119

Baba et al., 2006: 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

Brierley97: Brierley I, Meredith MR, Bloys AJ, Hagervall TG (1997). "Expression of a coronavirus ribosomal frameshift signal in Escherichia coli: influence of tRNA anticodon modification on frameshifting." J Mol Biol 270(3);360-73. PMID: 9237903

Dassain99: Dassain M, Leroy A, Colosetti L, Carole S, Bouche JP (1999). "A new essential gene of the 'minimal genome' affecting cell division." Biochimie 81(8-9);889-95. PMID: 10572302

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

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."

Hagervall98: Hagervall TG, Pomerantz SC, McCloskey JA (1998). "Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position." J Mol Biol 284(1);33-42. PMID: 9811540

Ikeuchi06: Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T (2006). "Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions." Mol Cell 21(1);97-108. PMID: 16387657

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

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

Kambampati03: Kambampati R, Lauhon CT (2003). "MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli." Biochemistry 42(4);1109-17. PMID: 12549933

Li07b: Li H (2007). "Complexes of tRNA and maturation enzymes: shaping up for translation." Curr Opin Struct Biol 17(3);293-301. PMID: 17580114

Nilsson02: Nilsson K, Lundgren HK, Hagervall TG, Bjork GR (2002). "The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium." J Bacteriol 184(24);6830-5. PMID: 12446633

Numata06a: Numata T, Ikeuchi Y, Fukai S, Adachi H, Matsumura H, Takano K, Murakami S, Inoue T, Mori Y, Sasaki T, Suzuki T, Nureki O (2006). "Crystallization and preliminary X-ray analysis of the tRNA thiolation enzyme MnmA from Escherichia coli complexed with tRNA(Glu)." Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 4);368-71. PMID: 16582487

Numata06b: Numata T, Ikeuchi Y, Fukai S, Suzuki T, Nureki O (2006). "Snapshots of tRNA sulphuration via an adenylated intermediate." Nature 442(7101);419-24. PMID: 16871210

Riley06: Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, Mori H, Perna NT, Plunkett G, Rudd KE, Serres MH, Thomas GH, Thomson NR, Wishart D, Wanner BL (2006). "Escherichia coli K-12: a cooperatively developed annotation snapshot--2005." Nucleic Acids Res 34(1);1-9. PMID: 16397293

Rogers95a: Rogers KC, Crescenzo AT, Soll D (1995). "Aminoacylation of transfer RNAs with 2-thiouridine derivatives in the wobble position of the anticodon." Biochimie 77(1-2);66-74. PMID: 7541255

Sullivan85a: Sullivan MA, Cannon JF, Webb FH, Bock RM (1985). "Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine." J Bacteriol 161(1);368-76. PMID: 3881393

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

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

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

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

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

Urbonavicius01: Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Bjork GR (2001). "Improvement of reading frame maintenance is a common function for several tRNA modifications." EMBO J 20(17);4863-73. PMID: 11532950

Urbonavicius03: Urbonavicius J, Stahl G, Durand JM, Ben Salem SN, Qian Q, Farabaugh PJ, Bjork GR (2003). "Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting." RNA 9(6);760-8. PMID: 12756333

Other References Related to Gene Regulation

Zaslaver06: Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, Alon U (2006). "A comprehensive library of fluorescent transcriptional reporters for Escherichia coli." Nat Methods 3(8);623-8. PMID: 16862137


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Tue Nov 25, 2014, BIOCYC14B.