|Gene:||rtcB||Accession Numbers: G7751 (EcoCyc), b3421, ECK3407|
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].
|Map Position: [3,554,875 <- 3,556,101] (76.62 centisomes, 276°)||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
|Biological Process:||GO:0006266 - DNA ligation
GO:0006281 - DNA repair [UniProtGOA11a, Das13]
GO:0006396 - RNA processing [GOA01a, Tanaka11]
GO:0006974 - cellular response to DNA damage stimulus [UniProtGOA11a, Das13]
GO:0042245 - RNA repair [Tanaka11, Tanaka11b]
|Molecular Function:||GO:0003909 - DNA ligase activity
GO:0008452 - RNA ligase activity [GOA01a, Tanaka11]
GO:0030145 - manganese ion binding [Das13, Tanaka11]
GO:0016874 - ligase activity [UniProtGOA11a]
GO:0046872 - metal ion binding [UniProtGOA11a]
|Cellular Component:||GO:0005829 - cytosol [DiazMejia09]|
|MultiFun Terms:||information transfer → DNA related|
|information transfer → RNA related|
|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]|
Enzymatic reaction of: RNA ligase
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
|Feature Class||Location||Common Name||Attached Group||Citations||Comment|
|Nucleotide-Phosphate-Binding-Region||281 -> 282||GMP|
|Nucleotide-Phosphate-Binding-Region||311 -> 314||GMP|
|Nucleotide-Phosphate-Binding-Region||337 -> 340||GMP|
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.
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
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
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
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
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