RlmN is the dual-specificity RNA methyltransferase of Escherichia coli that installs 2-methyladenosine at A2503 of 23S rRNA, in the peptidyl transferase centre at the entrance to the nascent peptide exit tunnel, and at A37 next to the anticodon of a subset of tRNAs. It is a radical S-adenosyl-L-methionine (SAM) enzyme with a single [4Fe-4S] cluster bound by a CxxxCxxC motif. Catalysis is a ping-pong reaction: one SAM methylates Cys355 by SN2 transfer, a second SAM is reductively cleaved to a 5'-deoxyadenosyl radical that abstracts a hydrogen from the Cys355 methyl, and the resulting methylene radical adds to C2 of the adenine to form a covalent protein-RNA intermediate that is resolved with the help of Cys118. RlmN acts on protein-free 23S rRNA, implying modification during ribosome assembly, and on nearly mature tRNA, unfolding the anticodon stem-loop to reach A37. Loss of RlmN abolishes m2A in both RNAs, reduces competitive fitness, increases UAG stop-codon read-through and slightly raises susceptibility to some peptidyl transferase inhibitors. Its paralog Cfr methylates C8 of the same adenosine and confers multidrug resistance.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000049 tRNA binding | IDA PMID:22891362 The Escherichia coli RlmN methyltransferase is a dual-specif... | KEEP AS NON CORE | Summary: tRNA binding: RlmN binds its tRNA substrates; the crystal structure of the cross-linked intermediate shows contacts along the whole tRNAGlu (PMID:27081063). Reason: Binding of the tRNA substrate is real but is a part of the tRNA (adenine(37)-C2)-methyltransferase activity, which is the informative core term. Kept as non-core substrate binding, not removed. Supporting Evidence: PMID:27081063 RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop PMID:22891362 Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. |
| GO:0000049 tRNA binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: tRNA binding: RlmN binds its tRNA substrates; the crystal structure of the cross-linked intermediate shows contacts along the whole tRNAGlu (PMID:27081063). Reason: Binding of the tRNA substrate is real but is a part of the tRNA (adenine(37)-C2)-methyltransferase activity, which is the informative core term. Kept as non-core substrate binding, not removed. Supporting Evidence: PMID:27081063 RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop PMID:22891362 Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. |
| GO:0002935 tRNA (adenine(37)-C2)-methyltransferase activity | IDA PMID:22891362 The Escherichia coli RlmN methyltransferase is a dual-specif... | ACCEPT | Summary: RlmN also methylates C2 of A37 in a subset of tRNAs. The deletion mutant lacks m2A in tRNA, plasmid expression restores it, and purified RlmN makes m2A37 on a tRNA chimera in vitro (PMID:22891362). Later work defined tRNA determinants (position 38; PMID:27902775) and captured RlmN cross-linked to tRNAGlu A37 (PMID:27081063). Reason: Second core activity of this dual-specificity enzyme, supported by genetic (IMP) and in vitro (IDA) evidence from the same study, the crystal structure of the tRNA-bound intermediate and the curated UniProt function text. The cached paper is abstract-only, but the abstract states both the knockout/rescue and the in vitro result. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. PMID:22891362 Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. PMID:27081063 RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop |
| GO:0002935 tRNA (adenine(37)-C2)-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RlmN also methylates C2 of A37 in a subset of tRNAs. The deletion mutant lacks m2A in tRNA, plasmid expression restores it, and purified RlmN makes m2A37 on a tRNA chimera in vitro (PMID:22891362). Later work defined tRNA determinants (position 38; PMID:27902775) and captured RlmN cross-linked to tRNAGlu A37 (PMID:27081063). Reason: Second core activity of this dual-specificity enzyme, supported by genetic (IMP) and in vitro (IDA) evidence from the same study, the crystal structure of the tRNA-bound intermediate and the curated UniProt function text. The cached paper is abstract-only, but the abstract states both the knockout/rescue and the in vitro result. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. PMID:22891362 Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. PMID:27081063 RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop |
| GO:0002935 tRNA (adenine(37)-C2)-methyltransferase activity | IMP PMID:22891362 The Escherichia coli RlmN methyltransferase is a dual-specif... | ACCEPT | Summary: RlmN also methylates C2 of A37 in a subset of tRNAs. The deletion mutant lacks m2A in tRNA, plasmid expression restores it, and purified RlmN makes m2A37 on a tRNA chimera in vitro (PMID:22891362). Later work defined tRNA determinants (position 38; PMID:27902775) and captured RlmN cross-linked to tRNAGlu A37 (PMID:27081063). Reason: Second core activity of this dual-specificity enzyme, supported by genetic (IMP) and in vitro (IDA) evidence from the same study, the crystal structure of the tRNA-bound intermediate and the curated UniProt function text. The cached paper is abstract-only, but the abstract states both the knockout/rescue and the in vitro result. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. PMID:22891362 Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. PMID:27081063 RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Catalytic activity, from the radical SAM domain (IPR007197). Reason: Per the RADICAL_SAM treatment rule, the root catalytic term is replaced by the specific reaction terms the gene carries, here the two C2-methyltransferase activities on 23S rRNA A2503 and tRNA A37. Proposed replacements: rRNA (adenine(2503)-C2-)-methyltransferase activity tRNA (adenine(37)-C2)-methyltransferase activity Supporting Evidence: file:ECOLI/rlmN/rlmN-uniprot.txt Belongs to the radical SAM superfamily. RlmN family. PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. |
| GO:0005515 protein binding | IPI PMID:15690043 Interaction network containing conserved and essential prote... | REMOVE | Summary: Protein binding with RplO (uL15), from TAP-MS affinity purification (PMID:15690043). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:15690043 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry |
| GO:0005515 protein binding | IPI PMID:15690043 Interaction network containing conserved and essential prote... | REMOVE | Summary: Protein binding with MalT, from TAP-MS affinity purification (PMID:15690043). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:15690043 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry |
| GO:0005515 protein binding | IPI PMID:15690043 Interaction network containing conserved and essential prote... | REMOVE | Summary: Protein binding with SrmB, from TAP-MS affinity purification (PMID:15690043). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:15690043 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Protein binding with RplO (uL15), from a genome-wide yeast two-hybrid screen (PMID:24561554). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:24561554 Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Protein binding with MalT, from a genome-wide yeast two-hybrid screen (PMID:24561554). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:24561554 Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Protein binding with FtsZ, from a genome-wide yeast two-hybrid screen (PMID:24561554). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:24561554 Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Protein binding with SrmB, from a genome-wide yeast two-hybrid screen (PMID:24561554). Reason: Generic protein binding is uninformative, and these high-throughput interactions do not support a more specific molecular function for RlmN. Removal does not mean the interaction is false. Co-purification with RplO (uL15) and SrmB (a 50S-assembly DEAD-box helicase) would fit RlmN acting on 23S rRNA during subunit assembly (PMID:20184321), but that is an inference about substrate access, not an activity of RlmN. Supporting Evidence: PMID:24561554 Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasm, from UniProt subcellular location and the HAMAP rule. Reason: RlmN is a soluble cytoplasmic enzyme acting on 23S rRNA before 50S assembly is complete and on tRNA; consistent with the cytosol IDA. Supporting Evidence: PMID:20184321 neither enzyme can act on the assembled 50S subunit or the 70S ribosome, but protein-free 23S rRNA proved to be a good substrate |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Cytosol, from the PANTHER node (IBA). Reason: Consistent with the cytosolic proteome IDA (PMID:18304323) and the soluble nature of the enzyme. Supporting Evidence: PMID:18304323 Here, we describe an approach to maximize MS based proteome identification coverage in an application to the E. coli cytosol |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Cytosol, from the E. coli cytosolic proteome abundance study (PMID:18304323). Reason: RlmN is a soluble enzyme with no membrane or secretion signals, and its substrates (free 23S rRNA during assembly, tRNA) are cytosolic. The protein list is in the paper's supplementary data, which is not in the cache; the curator's IDA is accepted. Supporting Evidence: PMID:18304323 Here, we describe an approach to maximize MS based proteome identification coverage in an application to the E. coli cytosol |
| GO:0006364 rRNA processing | IEA GO_REF:0000002 | MODIFY | Summary: rRNA processing, from the InterPro RlmN/Cfr family mapping (IPR004383). Reason: RlmN does not cleave or process rRNA; it methylates a base in 23S rRNA, so the correct process is rRNA base methylation (GO:0070475), which RlmN already carries by IMP. Same correction as for the paralog Cfr in genes/STAWA/cfr. Proposed replacements: rRNA base methylation Supporting Evidence: PMID:18025251 These result unequivocally established RlmN as the methyltransferase that modifies A2503 in 23S rRNA to m 2 A. |
| GO:0008173 RNA methyltransferase activity | IBA GO_REF:0000033 | MODIFY | Summary: RNA methyltransferase activity, from the PANTHER family node (IBA) and InterPro IPR004383 (IEA). Reason: True but general. RlmN is a dual-specificity enzyme with two specific, experimentally supported activities that it already carries: rRNA (adenine(2503)-C2-)-methyltransferase (GO:0070040) and tRNA (adenine(37)-C2)-methyltransferase (GO:0002935). This is a refinement, not a challenge to the PAINT node: both descendants entail the IBA claim. Proposed replacements: rRNA (adenine(2503)-C2-)-methyltransferase activity tRNA (adenine(37)-C2)-methyltransferase activity Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. file:ECOLI/rlmN/rlmN-uniprot.txt Specifically methylates position 2 of adenine 2503 in 23S rRNA and position 2 of adenine 37 in tRNAs. |
| GO:0008173 RNA methyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: RNA methyltransferase activity, from the PANTHER family node (IBA) and InterPro IPR004383 (IEA). Reason: True but general. RlmN is a dual-specificity enzyme with two specific, experimentally supported activities that it already carries: rRNA (adenine(2503)-C2-)-methyltransferase (GO:0070040) and tRNA (adenine(37)-C2)-methyltransferase (GO:0002935). This is a refinement, not a challenge to the PAINT node: both descendants entail the IBA claim. Proposed replacements: rRNA (adenine(2503)-C2-)-methyltransferase activity tRNA (adenine(37)-C2)-methyltransferase activity Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. file:ECOLI/rlmN/rlmN-uniprot.txt Specifically methylates position 2 of adenine 2503 in 23S rRNA and position 2 of adenine 37 in tRNAs. |
| GO:0019843 rRNA binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: rRNA binding, from the HAMAP RlmN family rule. Reason: RlmN must bind 23S rRNA (helices 90-92 and the A2503 region of protein-free 23S rRNA) to methylate it (PMID:20184321), but this is substrate binding entailed by the rRNA methyltransferase activity. Kept as non-core, consistent with the treatment of rRNA binding in genes/STAWA/cfr. Supporting Evidence: PMID:20184321 neither enzyme can act on the assembled 50S subunit or the 70S ribosome, but protein-free 23S rRNA proved to be a good substrate |
| GO:0030488 tRNA methylation | IDA PMID:22891362 The Escherichia coli RlmN methyltransferase is a dual-specif... | ACCEPT | Summary: tRNA methylation: RlmN installs m2A37 in tRNA (PMID:22891362). Reason: RlmN itself catalyses the methylation step, so it participates directly. GO has no more specific child for adenine C2 methylation in tRNA (children of GO:0030488 checked in QuickGO cover ribose, N1/N2/N7-guanine and C5/C3-cytosine methylation only), so this term is as precise as the ontology allows. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. |
| GO:0030488 tRNA methylation | IEA GO_REF:0000120 | ACCEPT | Summary: tRNA methylation: RlmN installs m2A37 in tRNA (PMID:22891362). Reason: RlmN itself catalyses the methylation step, so it participates directly. GO has no more specific child for adenine C2 methylation in tRNA (children of GO:0030488 checked in QuickGO cover ribose, N1/N2/N7-guanine and C5/C3-cytosine methylation only), so this term is as precise as the ontology allows. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. |
| GO:0030488 tRNA methylation | IMP PMID:22891362 The Escherichia coli RlmN methyltransferase is a dual-specif... | ACCEPT | Summary: tRNA methylation: RlmN installs m2A37 in tRNA (PMID:22891362). Reason: RlmN itself catalyses the methylation step, so it participates directly. GO has no more specific child for adenine C2 methylation in tRNA (children of GO:0030488 checked in QuickGO cover ribose, N1/N2/N7-guanine and C5/C3-cytosine methylation only), so this term is as precise as the ontology allows. Supporting Evidence: PMID:22891362 Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. PMID:22891362 The ΞrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Iron-sulfur cluster binding, from the radical SAM domain (IPR007197). Reason: Correct but general. RlmN binds a single [4Fe-4S] cluster through the CxxxCxxC motif (PMID:21916495, PMID:20184321), so the specific child GO:0051539, which RlmN already carries by IDA, is the right term. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:21916495 Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor file:ECOLI/rlmN/rlmN-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:20184321 RlmN and Cfr are radical SAM enzymes involved in methylation... | ACCEPT | Summary: RlmN binds one radical SAM [4Fe-4S] cluster, ligated by Cys125, Cys129 and Cys132 with SAM at the unique iron (PMID:20184321, PMID:21916495, PMID:21527678). Reason: The radical SAM cluster is the catalytic cofactor and is required for both SAM reactivities (Cys355 methylation and 5'-dA radical generation), so it is core. Cluster-free RlmN does not methylate Cys355 (PMID:21916495). Supporting Evidence: PMID:20184321 We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. PMID:21916495 Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor PMID:21527678 Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000104 | ACCEPT | Summary: RlmN binds one radical SAM [4Fe-4S] cluster, ligated by Cys125, Cys129 and Cys132 with SAM at the unique iron (PMID:20184321, PMID:21916495, PMID:21527678). Reason: The radical SAM cluster is the catalytic cofactor and is required for both SAM reactivities (Cys355 methylation and 5'-dA radical generation), so it is core. Cluster-free RlmN does not methylate Cys355 (PMID:21916495). Supporting Evidence: PMID:20184321 We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. PMID:21916495 Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor PMID:21527678 Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. |
| GO:0070040 rRNA (adenine(2503)-C2-)-methyltransferase activity | IDA PMID:20184321 RlmN and Cfr are radical SAM enzymes involved in methylation... | ACCEPT | Summary: RlmN methylates C2 of A2503 in 23S rRNA, forming m2A2503. Knockout loses m2A2503 and plasmid rescue restores it (PMID:18025251); the reconstituted enzyme methylates protein-free 23S rRNA at C2 in vitro using SAM as methyl donor and radical source (PMID:20184321). Reason: This is the founding activity of RlmN, established genetically (IMP, PMID:18025251), biochemically (IDA, PMID:20184321) and mechanistically (PMID:21415317, PMID:21527678). The GO term definition is the radical SAM reaction itself. The IEA row from the HAMAP/Rhea mapping agrees. Supporting Evidence: PMID:18025251 These result unequivocally established RlmN as the methyltransferase that modifies A2503 in 23S rRNA to m 2 A. PMID:20184321 These experiments showed that while RlmN catalyzes methylation at position C2 of the substrate PMID:20184321 S-Adenosylmethionine (SAM) is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. file:ECOLI/rlmN/rlmN-uniprot.txt Specifically methylates position 2 of adenine 2503 in 23S rRNA and position 2 of adenine 37 in tRNAs. |
| GO:0070040 rRNA (adenine(2503)-C2-)-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RlmN methylates C2 of A2503 in 23S rRNA, forming m2A2503. Knockout loses m2A2503 and plasmid rescue restores it (PMID:18025251); the reconstituted enzyme methylates protein-free 23S rRNA at C2 in vitro using SAM as methyl donor and radical source (PMID:20184321). Reason: This is the founding activity of RlmN, established genetically (IMP, PMID:18025251), biochemically (IDA, PMID:20184321) and mechanistically (PMID:21415317, PMID:21527678). The GO term definition is the radical SAM reaction itself. The IEA row from the HAMAP/Rhea mapping agrees. Supporting Evidence: PMID:18025251 These result unequivocally established RlmN as the methyltransferase that modifies A2503 in 23S rRNA to m 2 A. PMID:20184321 These experiments showed that while RlmN catalyzes methylation at position C2 of the substrate PMID:20184321 S-Adenosylmethionine (SAM) is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. file:ECOLI/rlmN/rlmN-uniprot.txt Specifically methylates position 2 of adenine 2503 in 23S rRNA and position 2 of adenine 37 in tRNAs. |
| GO:0070040 rRNA (adenine(2503)-C2-)-methyltransferase activity | IMP PMID:18025251 The methyltransferase YfgB/RlmN is responsible for modificat... | ACCEPT | Summary: RlmN methylates C2 of A2503 in 23S rRNA, forming m2A2503. Knockout loses m2A2503 and plasmid rescue restores it (PMID:18025251); the reconstituted enzyme methylates protein-free 23S rRNA at C2 in vitro using SAM as methyl donor and radical source (PMID:20184321). Reason: This is the founding activity of RlmN, established genetically (IMP, PMID:18025251), biochemically (IDA, PMID:20184321) and mechanistically (PMID:21415317, PMID:21527678). The GO term definition is the radical SAM reaction itself. The IEA row from the HAMAP/Rhea mapping agrees. Supporting Evidence: PMID:18025251 Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA PMID:18025251 These result unequivocally established RlmN as the methyltransferase that modifies A2503 in 23S rRNA to m 2 A. |
| GO:0070475 rRNA base methylation | IEA GO_REF:0000120 | ACCEPT | Summary: rRNA base methylation: RlmN methylates the adenine base of A2503 in 23S rRNA (PMID:18025251, PMID:20184321). Reason: Correct and appropriately specific process for the C2 methylation of the A2503 base; RlmN catalyses the step. Matches the process used for the paralog Cfr (genes/STAWA/cfr). Supporting Evidence: PMID:18025251 Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA PMID:20184321 These experiments showed that while RlmN catalyzes methylation at position C2 of the substrate |
| GO:0070475 rRNA base methylation | IMP PMID:18025251 The methyltransferase YfgB/RlmN is responsible for modificat... | ACCEPT | Summary: rRNA base methylation: RlmN methylates the adenine base of A2503 in 23S rRNA (PMID:18025251, PMID:20184321). Reason: Correct and appropriately specific process for the C2 methylation of the A2503 base; RlmN catalyses the step. Matches the process used for the paralog Cfr (genes/STAWA/cfr). Supporting Evidence: PMID:18025251 Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA PMID:20184321 These experiments showed that while RlmN catalyzes methylation at position C2 of the substrate |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:21527678 Structural basis for methyl transfer by a radical SAM enzyme... | NEW | Summary: RlmN binds SAM at the unique iron of its [4Fe-4S] cluster; crystal structures of RlmN with SAM show one SAM coordinated to the cluster, next to the S-methylated Cys355 (PMID:21527678). Reason: GOA has no SAM-binding row for RlmN, although SAM is the cosubstrate of both half-reactions (Cys355 methylation by SN2 and 5'-dA radical generation; PMID:21916495). This is a molecular function of RlmN itself, shown directly by the SAM-bound crystal structure, so participation is not in question. Matches the RADICAL_SAM project convention for every member with established radical SAM catalysis (e.g. LIAS, CDKAL1). Supporting Evidence: PMID:21527678 Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. PMID:21916495 SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities file:ECOLI/rlmN/rlmN-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
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Download this section (compressed HTML)Q: Is the higher UAG read-through of the rlmN deletion caused directly by loss of m2A2503 at the peptidyl transferase centre, or partly by loss of m2A37 in tRNA?
Q: How does one RlmN active site recognise two structurally different substrates (the A2503 region of 23S rRNA and the tRNA anticodon loop), and does it modify 23S rRNA at a defined stage of 50S assembly in vivo?
Experiment: Measure stop-codon read-through and missense reporters in strains whose 23S rRNA carries an A2503G substitution, or that express RlmN variants which keep tRNA activity but lose rRNA activity, and compare them with the full deletion.
Hypothesis: The fidelity phenotype comes from the rRNA modification, not the tRNA modification.
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