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Escherichia coli K-12 substr. MG1655 Enzyme: RNA ligase



Gene: rtcB Accession Numbers: G7751 (EcoCyc), b3421, ECK3407

Synonyms: yhgL

Regulation Summary Diagram: ?

Summary:
RtcB is a GTP-dependent 3'-5' RNA ligase with the ability to join RNA 2',3'-cyclic-PO4 or 3'-PO4 ends to RNA 5'-OH ends [Tanaka11, Tanaka11a, Desai12, Chakravarty12]. RtcB can perform RNA repair and tRNA splicing functions when expressed in yeast host cells [Tanaka11b].

RtcB can ligate 3'-PO4 and 5'-OH DNA ends that contain a single ribonucleoside 3'-PO4 or a 2',3'-cyclic-PO4, and can perform a DNA splicing reaction utilizing DNA 3'-PO4 and 5'-OH ends via a DNA3'pp5'G intermediate. This is a novel activity; "classic" DNA ligases are unable to ligate such substrates [Das13].

Conserved residues that are essential for RtcB activity have been identified by site-directed mutagenesis [Tanaka11b, Chakravarty12]. The reaction appears to follow a three-step mechanism with initial activation of RtcB by GTP hydrolysis and formation of a covalent RtcB-guanylate adduct [Tanaka11a, Desai12, Chakravarty12]. The guanylate group is then transferred to the 3'-phosphate of a polynucleotide, forming a polynucleotide-(3')pp(5')G intermediate. A 5'-OH then attacks the -N(3')pp(5')G intermediate, forming the ligated product [Chakravarty12]. An alternative hypothesis involves generation of a 2'-3' cyclic phosphate intermediate and release of GMP followed by the attack of a 5'-OH of a second RNA and subsequent ligation to the 3' phosphate [Desai12]. Further experiments indicate that the activated RNA(3')pp(5')G is an obligate intermediate even during ligation of a substrate that already contains the 2'-3' cyclic phosphate end, arguing against this alternative reaction mechanism [Chakravarty12a].

Expression of the rtcBA operon is regulated by RtcR from a σ54-dependent promoter [Genschik98].

Locations: cytosol

Map Position: [3,554,875 <- 3,556,101] (76.62 centisomes)
Length: 1227 bp / 408 aa

Molecular Weight of Polypeptide: 45.222 kD (from nucleotide sequence), 45.0 kD (experimental) [Tanaka11 ]

Unification Links: ASAP:ABE-0011170 , EchoBASE:EB2774 , EcoGene:EG12939 , EcoliWiki:b3421 , ModBase:P46850 , OU-Microarray:b3421 , PortEco:rtcB , PR:PRO_000023892 , Protein Model Portal:P46850 , RefSeq:NP_417879 , RegulonDB:G7751 , SMR:P46850 , String:511145.b3421 , UniProt:P46850

Relationship Links: InterPro:IN-FAMILY:IPR001233 , Panther:IN-FAMILY:PTHR11118 , Pfam:IN-FAMILY:PF01139 , Prosite:IN-FAMILY:PS01288

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006266 - DNA ligation Inferred from experiment [Das13]
GO:0006281 - DNA repair Inferred from experiment [Das13]
GO:0006396 - RNA processing Inferred from experiment Inferred by computational analysis [GOA01, Tanaka11]
GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Das13]
GO:0042245 - RNA repair Inferred from experiment Author statement [Tanaka11, Tanaka11b]
Molecular Function: GO:0003909 - DNA ligase activity Inferred from experiment [Das13]
GO:0008452 - RNA ligase activity Inferred from experiment Inferred by computational analysis [GOA01, Tanaka11]
GO:0030145 - manganese ion binding Inferred from experiment [Das13, Tanaka11]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: information transfer DNA related
information transfer RNA related

Essentiality data for rtcB knockouts: ?

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

Credits:
Last-Curated ? 21-Nov-2013 by Keseler I , SRI International


Enzymatic reaction of: RNA ligase

an RNA terminal-2',3'-cyclic-phosphate + a 5'-hydroxyl terminated RNA + GTP <=> a ligated RNA + GMP + 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 an RNA terminal-2',3'-cyclic-phosphate: an RNA 3'-terminal-phosphate

Cofactors or Prosthetic Groups: Mn2+ [Tanaka11a, Tanaka11]

Kinetic Parameters:

Substrate
Km (μM)
Citations
GTP
16.0
[Desai12]


Sequence Features

Feature Class Location Common Name Citations Comment
Metal-Binding-Site 78  
[UniProt11]
UniProt: Zinc; Non-Experimental Qualifier: potential.
Sequence-Conflict 84  
[Blattner97, UniProt11]
Alternate sequence: R → G; UniProt: (in Ref. 1; AAA58219).
Metal-Binding-Site 168  
[UniProt11]
UniProt: Zinc; Non-Experimental Qualifier: potential.
Metal-Binding-Site 185  
[UniProt11]
UniProt: Zinc; Non-Experimental Qualifier: potential.
Modified-Residue 337 (histidinyl-N)-GMP
[Chakravarty12, Chakravarty12]
 


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


References

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

Blattner97: Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997). "The complete genome sequence of Escherichia coli K-12." Science 277(5331);1453-74. PMID: 9278503

Chakravarty12: Chakravarty AK, Subbotin R, Chait BT, Shuman S (2012). "RNA ligase RtcB splices 3'-phosphate and 5'-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3')pp(5')G intermediates." Proc Natl Acad Sci U S A 109(16);6072-7. PMID: 22474365

Chakravarty12a: Chakravarty AK, Shuman S (2012). "The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent." Nucleic Acids Res 40(17);8558-67. PMID: 22730297

Das13: Das U, Chakravarty AK, Remus BS, Shuman S (2013). "Rewriting the rules for end joining via enzymatic splicing of DNA 3'-PO4 and 5'-OH ends." Proc Natl Acad Sci U S A 110(51);20437-42. PMID: 24218597

Desai12: Desai KK, Raines RT (2012). "tRNA ligase catalyzes the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini." Biochemistry 51(7);1333-5. PMID: 22320833

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

Genschik98: Genschik P, Drabikowski K, Filipowicz W (1998). "Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its sigma54-regulated operon." J Biol Chem 273(39);25516-26. PMID: 9738023

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."

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

Tanaka11: Tanaka N, Shuman S (2011). "RtcB is the RNA ligase component of an Escherichia coli RNA repair operon." J Biol Chem 286(10);7727-31. PMID: 21224389

Tanaka11a: Tanaka N, Chakravarty AK, Maughan B, Shuman S (2011). "Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-Phosphate/5'-hydroxyl ligation reactions." J Biol Chem 286(50);43134-43. PMID: 22045815

Tanaka11b: Tanaka N, Meineke B, Shuman S (2011). "RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo." J Biol Chem 286(35);30253-7. PMID: 21757685

UniProt11: 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 manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."


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 Wed Nov 26, 2014, biocyc13.