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Information on EC 6.5.1.8 - 3'-phosphate/5'-hydroxy nucleic acid ligase and Organism(s) Escherichia coli and UniProt Accession P46850
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6.5.1.8 3'-phosphate/5'-hydroxy nucleic acid ligaseIUBMB Comments
The enzyme is a GTP- and Mn2+-dependent 3'-5' nucleic acid ligase with the ability to join RNA with 3'-phosphate or 2',3'-cyclic-phosphate ends to RNA with 5'-hydroxy ends. It can also join DNA with 3'-phosphate ends to DNA with 5'-hydroxy ends, provided the DNA termini are unpaired . The enzyme is found in members of all three kingdoms of life, and is essential in metazoa for the splicing of intron-containing tRNAs. The reaction follows a three-step mechanism with initial activation of the enzyme by GTP hydrolysis, forming a phosphoramide bond between the guanylate and a histidine residue. The guanylate group is transferred to the 3'-phosphate terminus of the substrate, forming the capped structure [DNA/RNA]-3'-(5'-diphosphoguanosine). When a suitable 5'-OH end is available, the enzyme catalyses an attack of the 5'-OH on the capped end to form a 3'-5' phosphodiester splice junction, releasing the guanylate. When acting on an RNA 2',3'-cyclic-phosphate, the enzyme catalyses an additional reaction, hydrolysing the cyclic phosphate to a 3'-phosphate . The metazoan enzyme requires activating cofactors in order to achieve multiple turnover catalysis .
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
Reaction Schemes
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5'-hydroxy-(ribonucleotide)m
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(ribonucleotide)n-2',3'-cyclophosphate
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5'-hydroxy-(ribonucleotide)m
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Synonyms
rtcb-1, rtcb1, rtcb rna ligase, trna-splicing ligase, rna-splicing ligase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
rtcB
rtcB
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
poly(ribonucleotide)-3'-phosphate:5'-hydroxy-poly(ribonucleotide) ligase (GMP-forming)
The enzyme is a GTP- and Mn2+-dependent 3'-5' nucleic acid ligase with the ability to join RNA with 3'-phosphate or 2',3'-cyclic-phosphate ends to RNA with 5'-hydroxy ends. It can also join DNA with 3'-phosphate ends to DNA with 5'-hydroxy ends, provided the DNA termini are unpaired [6]. The enzyme is found in members of all three kingdoms of life, and is essential in metazoa for the splicing of intron-containing tRNAs. The reaction follows a three-step mechanism with initial activation of the enzyme by GTP hydrolysis, forming a phosphoramide bond between the guanylate and a histidine residue. The guanylate group is transferred to the 3'-phosphate terminus of the substrate, forming the capped structure [DNA/RNA]-3'-(5'-diphosphoguanosine). When a suitable 5'-OH end is available, the enzyme catalyses an attack of the 5'-OH on the capped end to form a 3'-5' phosphodiester splice junction, releasing the guanylate. When acting on an RNA 2',3'-cyclic-phosphate, the enzyme catalyses an additional reaction, hydrolysing the cyclic phosphate to a 3'-phosphate [9]. The metazoan enzyme requires activating cofactors in order to achieve multiple turnover catalysis [8].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
(ribonucleotide)n-2',3'-cyclophosphate + H2O
(ribonucleotide)n-3'-phosphate
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?
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + 5'-hydroxy-(ribonucleotide)m
(ribonucleotide)n+m + GMP
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(ribonucleotide)n-3'-phosphate + 5'-hydroxy-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
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overall reaction, substrate 5'-hydroxy-(ribonucleotide)m can be composed of all ribonucleotides or all deoxynucleotides
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(ribonucleotide)n-3'-phosphate + 5'-phospho-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
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overall reaction
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5'-guanosyl [RNA ligase]-NT-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
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RtcB transfers GMP to the RNA 3'-phosphate end
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5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
GTP + [RNA ligase]-L-histidine
5'-guanosyl [RNA ligase]-NT-phosphono-L-histidine + diphosphate
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GTP + [RNA ligase]-L-histidine
5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + diphosphate
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(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
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(ribonucleotide)n-3'-phosphate + 5'-hydroxy-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
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(truncated 16S rRNA)-3'-phosphate + 5'-hydroxy-RNA43 + GTP
16S rRNA + GMP + diphosphate
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endoribonuclease MazF, the toxin component of the toxin-antitoxin module mazEF, specifically cleaves the 16S rRNA of 70S ribosomes at an ACA site located at positions 15001502. Thereby, the ribosome loses a 3'-terminal 16S rRNA fragment of 43 nucleotides. RNA ligase RtcB catalyzes the religation of the truncated 16S rRNA present in specialized ribosomes. Thereby their ability to translate canonical mRNAs is fully restored
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(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
substrate for ligation is a 20mer RNA strand with 5'-OH and 2',3'-cyclic phosphate ends
overall reaction
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?
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
substrate mimicks the broken tRNAGlu(UUC) anticodon stem-loop generated by Kluyveromyces lactis gamma-toxin, leaving 2',3'-cyclic phosphate and 5'-OH ends
overall reaction
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?
5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
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5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
substrate for ligation is a 20-mer RNA strand with 5'-OH and 3'-monophosphate ends
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additional information
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RtcB executes a three-step ligation pathway, consisting of reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate, transfer of guanylate to a polynucleotide 3'-phosphate to form a polynucleotide-(3')pp(5')G intermediate, and (iii) attack of a 5'-OH on the N(3')pp(5')G end to form the splice junction. Presence of GTP is required
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additional information
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RtcB seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester
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additional information
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sealing of a 2',3'-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcBGMP adduct, and involves a kinetically valid RNA(3')pp(5')G intermediate. RtcB catalyzes the hydrolysis of a 2',3'-cyclic phosphate to a 3'-phosphate at a rate that is at least as fast as the rate of ligation. NTP requirement for ligation is satisfied by GTP and 6-O-methyl guanosine triphosphate and inosine triphosphate. 6-chloropurine ribonucleoside triphosphate, 2-aminopurine ribonucleoside triphosphate and ATP are inactive
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additional information
?
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the enzyme joins RNA 2',3'-cyclic phosphate or RNA 3'-phosphate ends to 5'-OH RNA ends in a multistep pathway whereby the enzyme hydrolyzes RNA 2',3'-cyclic phosphate to RNA 3'-phosphate, transfers GMP from GTP to RNA 3'-phosphate to form to RNAppG, and directs the attack of 5'-OH on RNAppG to form a 3'-5' phosphodiester splice junction
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
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?
(ribonucleotide)n-3'-phosphate + 5'-hydroxy-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mn2+
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
physiological function
RtcB guanylylation precedes the cyclic phosphodiesterase and 3'-phosphate ligase steps of the RNA splicing pathway
physiological function
RtcB is able in vivo to complement growth of yeast cells that lack the endogenous healing/sealing-type tRNA ligase Trl1. RtcB also protects yeast Trl1 mutant cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response
physiological function
RtcB ligates 3'-monophosphate and 5'-OH ends and has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP
physiological function
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upon ER stress, HAC1/XBP1 undergoes exon/intron-specific excision by inositol requiring enzyme 1 to remove an intron and liberate the 5' and 3' exons, which are ligated by tRNA ligase. Expression of RtcB in a Saccharomyces cerevisiae tRNA ligase mutant strain is able to complement HAC1/XBP1 splicing
physiological function
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the enzyme operates during the unfolded protein response
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H280A
active, protein is able to complement a Saccharomyces cerevisiae Trl1 mutant
H337A
H337N
H337Q
R189A
D75A
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the mutation allows cyclic phosphodiester hydrolysis but cripples 3'-phosphate guanylylation
H168A
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the mutant shows about 48% of 5'-OH-RNAp ligation activity compared to the wild type enzyme
H185A
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the mutant shows 5'-OH-RNAp ligation activity similar to the wild type enzyme
H281A
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the mutant is impaired in overall 5'-OH-RNAp and 5'-OH-RNA-2',3'-cyclic phosphate ligation but is able to seal a preguanylylated substrate
H337A
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the mutant shows about 1% of 5'-OH-RNAp ligation activity compared to the wild type enzyme
K299A
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the mutant shows about 50% of 5'-OH-RNAp ligation activity compared to the wild type enzyme
N167A
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the mutant shows about 65% of 5'-OH-RNAp ligation activity compared to the wild type enzyme
R189A
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the mutation slows the step of RNAppG/5'-OH RNA sealing by a factor of 200 compared to that with wild type enzyme while decreasing the rate of RNAppG formation by 3fold
R341A
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the mutant shows about 10% of 5'-OH-RNAp ligation activity compared to the wild type enzyme
R345A
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the mutant shows 5'-OH-RNAp ligation activity similar to the wild type enzyme
H337A
His337 is the site covalent guanylylation of RtcB, mutant is inactive
H337A
residue His337 is the site of covalent guanylylation, the mutation abolishes 3'-phosphate/5'-OH RNA ligation
H337N
residue His337 is the site of covalent guanylylation, the mutation abolishes 3'-phosphate/5'-OH RNA ligation
H337Q
residue His337 is the site of covalent guanylylation, the mutation abolishes 3'-phosphate/5'-OH RNA ligation
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
RtcB mRNA is processed by endoribonuclease MazF and selectively translated during stress
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Tanaka, N.; Shuman, S.
RtcB is the RNA ligase component of an Escherichia coli RNA repair operon
J. Biol. Chem.
286
7727-7731
2011
Escherichia coli (P46850)
Poothong, J.; Tirasophon, W.; Kaufman, R.
Functional analysis of the mammalian RNA ligase for IRE1 in the unfolded protein response
Biosci. Rep.
37
BSR20160574
2017
Escherichia coli, Homo sapiens (Q9Y3I0)
Tanaka, N.; Meineke, B.; Shuman, S.
RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo
J. Biol. Chem.
286
30253-30257
2011
Escherichia coli (P46850)
Tanaka, N.; Chakravarty, A.K.; Maughan, B.; Shuman, S.
Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-hydroxyl ligation reactions
J. Biol. Chem.
286
43134-43143
2011
Escherichia coli (P46850)
Chakravarty, A.K.; Shuman, S.
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
8558-8567
2012
Escherichia coli (P46850)
Temmel, H.; Mueller, C.; Sauert, M.; Vesper, O.; Reiss, A.; Popow, J.; Martinez, J.; Moll, I.
The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
Nucleic Acids Res.
45
4708-4721
2017
Escherichia coli
Chakravarty, A.K.; Subbotin, R.; Chait, B.T.; Shuman, S.
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. USA
109
6072-6077
2012
Escherichia coli (P46850)
Maughan, W.; Shuman, S.
Distinct contributions of enzymic functional groups to the 2',3'-cyclic phosphodiesterase, 3'-phosphate guanylylation, and 3'-ppG/5'-OH ligation steps of the Escherichia coli RtcB nucleic acid splicing pathway
J. Bacteriol.
198
1294-1304
2016
Escherichia coli
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