RimO is the radical S-adenosyl-L-methionine (SAM) methylthiotransferase of Escherichia coli that attaches a methylthio group to the beta-carbon of the universally conserved Asp88 (Asp89 counting the initiator methionine) of the small-subunit ribosomal protein uS12 (S12). It is the only known enzyme that methylthiolates a protein; the other members of the methylthiotransferase family, such as MiaB, modify tRNA. Despite this, RimO keeps the family's domain architecture: an N-terminal MTTase domain, a radical SAM domain and a C-terminal TRAM domain. It binds two [4Fe-4S] clusters, a radical SAM cluster that cleaves SAM to a 5'-deoxyadenosyl radical and an auxiliary cluster that activates an exogenous sulfur cosubstrate; by analogy with MiaB, a second SAM is thought to supply the methyl group. RimO is cytosolic, and modification appears to occur on uS12 assembled into a ribosomal subunit; the YcaO protein is needed for efficient modification. rimO mutants lack uS12 methylthiolation and show altered transcription of a subset of genes. The function of the modification, which sits near the ribosome decoding center, is not known.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro radical SAM signature mapping to the root catalytic activity term. Reason: True but uninformative. Per the RADICAL_SAM treatment rule, refined to the specific reaction RimO catalyses, protein (uS12 Asp89) methylthiolation, GO:0103039 (RHEA:37087, EC 2.8.4.4), which the gene already carries. Proposed replacements: protein methylthiotransferase activity Supporting Evidence: PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. file:ECOLI/rimO/rimO-uniprot.txt Catalyzes the methylthiolation of the residue Asp-89 of ribosomal protein uS12. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | MODIFY | Summary: UniProt/UniRule subcellular location: cytoplasm. Reason: Correct but general. RimO is a soluble enzyme detected in the E. coli cytosolic fraction and acting on uS12 in (pre)ribosomal particles; the gene already carries cytosol (GO:0005829) by IDA and IBA. Proposed replacements: cytosol Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228840 (RimO subfamily) is_active_in cytosol. Reason: Consistent with direct cytosolic detection; RimO modifies uS12, apparently in the context of a ribosomal subunit, in the cytosol. The target in WITH/FROM is its own experimental grounding. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 PMID:21169565 the proteomic data from this study provides direct evidence that the E. coli specific Ξ²-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit. |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Detected in the cytosolic fraction of E. coli MC4100 by LC-MS/MS abundance profiling. Reason: Direct (high-throughput) evidence for cytosolic location, consistent with RimO being a soluble protein-modifying enzyme without membrane or export signals. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0006400 tRNA modification | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from the MTTase family signature IPR005839 to tRNA modification. Reason: Demonstrably wrong for this protein. IPR005839 covers the whole methylthiotransferase family, whose other subfamilies (MiaB, MtaB, CDKAL1) modify tRNA, and the InterPro2GO mapping carries the tRNA process to every member. RimO is the one subfamily that modifies a protein: its substrate is Asp88 of ribosomal protein uS12, and it is the only known enzyme that methylthiolates a protein. There is no evidence that RimO modifies any tRNA. The narrower RimO signature (IPR005840) and the RimO-specific PAINT node PTN000228840 carry no tRNA term. Contrast miaB, where the same mapping is correct and is refined rather than removed. Supporting Evidence: PMID:19736993 To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. PMID:18252828 we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA PMID:23542644 The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. |
| GO:0016740 transferase activity | IEA GO_REF:0000120 | MODIFY | Summary: UniRule/InterPro mapping to the root transferase activity term. Reason: True but general; refined to the specific activity, protein methylthiotransferase activity (GO:0103039), and aspartic acid methylthiotransferase activity (GO:0035599), both descendants the gene already carries. Same treatment of GO:0016740 as in the CDK5RAP1, CDKAL1 and miaB reviews. Proposed replacements: protein methylthiotransferase activity aspartic acid methylthiotransferase activity Supporting Evidence: PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. |
| GO:0035596 methylthiotransferase activity | IEA GO_REF:0000120 | MODIFY | Summary: ARBA/InterPro mapping to methylthiotransferase activity. Reason: Correct but general. Both supported descendants are listed: GO:0103039 (uS12 Asp89 reaction) and GO:0035599 (peptidyl-Asp beta-methylthiolation), which the gene already carries. Proposed replacements: protein methylthiotransferase activity aspartic acid methylthiotransferase activity Supporting Evidence: PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. file:ECOLI/rimO/rimO-uniprot.txt Belongs to the methylthiotransferase family. RimO subfamily. |
| GO:0035599 aspartic acid methylthiotransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228840 (RimO subfamily); E. coli RimO is the experimental donor. Reason: Core molecular function. RimO methylthiolates the beta-carbon of Asp88 (UniProt numbering Asp89 with the initiator Met) of ribosomal protein uS12: the modification is absent in yliG/rimO mutants (PMID:18252828, PMID:21169565) and purified E. coli RimO methylthiolates an Asp88-loop peptide in vitro (PMID:19736993). GO:0035599 (peptidyl-aspartic acid beta-methylthiolation) and GO:0103039 (the RHEA:37087 uS12 Asp89 reaction) describe the same activity at slightly different granularity; they are siblings in the current ontology, not ancestor and descendant, so this row is accepted alongside GO:0103039 and is not TOO_GENERAL. The IBA node is the RimO subfamily, and E. coli RimO is its experimentally characterised member. Supporting Evidence: PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. file:ECOLI/rimO/rimO-uniprot.txt Belongs to the methylthiotransferase family. RimO subfamily. |
| GO:0035599 aspartic acid methylthiotransferase activity | IDA PMID:19736993 Characterization of RimO, a new member of the methylthiotran... | ACCEPT | Summary: Reconstituted E. coli RimO methylthiolates a peptide mimicking the uS12 Asp88 loop (MS detection), with reductive SAM cleavage to 5'-dA. Reason: Core molecular function. RimO methylthiolates the beta-carbon of Asp88 (UniProt numbering Asp89 with the initiator Met) of ribosomal protein uS12: the modification is absent in yliG/rimO mutants (PMID:18252828, PMID:21169565) and purified E. coli RimO methylthiolates an Asp88-loop peptide in vitro (PMID:19736993). GO:0035599 (peptidyl-aspartic acid beta-methylthiolation) and GO:0103039 (the RHEA:37087 uS12 Asp89 reaction) describe the same activity at slightly different granularity; they are siblings in the current ontology, not ancestor and descendant, so this row is accepted alongside GO:0103039 and is not TOO_GENERAL. Supporting Evidence: PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. PMID:19736993 Results from the HPLC-based kinetic assays imply that the reductive cleavage of SAM by RimOrcn to generate 5β²-dA takes place in the presence of the substrate analog P1. |
| GO:0035599 aspartic acid methylthiotransferase activity | IMP PMID:18252828 RimO, a MiaB-like enzyme, methylthiolates the universally co... | ACCEPT | Summary: Mass spectrometry of uS12 from a yliG mutant shows loss of Asp88 methylthiolation; yliG named rimO. Reason: Core molecular function. RimO methylthiolates the beta-carbon of Asp88 (UniProt numbering Asp89 with the initiator Met) of ribosomal protein uS12: the modification is absent in yliG/rimO mutants (PMID:18252828, PMID:21169565) and purified E. coli RimO methylthiolates an Asp88-loop peptide in vitro (PMID:19736993). GO:0035599 (peptidyl-aspartic acid beta-methylthiolation) and GO:0103039 (the RHEA:37087 uS12 Asp89 reaction) describe the same activity at slightly different granularity; they are siblings in the current ontology, not ancestor and descendant, so this row is accepted alongside GO:0103039 and is not TOO_GENERAL. Supporting Evidence: PMID:18252828 Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. |
| GO:0035599 aspartic acid methylthiotransferase activity | IMP PMID:21169565 A proteomic and transcriptomic approach reveals new insight ... | ACCEPT | Summary: Quantitative MS of uS12 in rimO and ycaO deletion strains: loss of rimO abolishes uS12 beta-methylthiolation. Reason: Core molecular function. RimO methylthiolates the beta-carbon of Asp88 (UniProt numbering Asp89 with the initiator Met) of ribosomal protein uS12: the modification is absent in yliG/rimO mutants (PMID:18252828, PMID:21169565) and purified E. coli RimO methylthiolates an Asp88-loop peptide in vitro (PMID:19736993). GO:0035599 (peptidyl-aspartic acid beta-methylthiolation) and GO:0103039 (the RHEA:37087 uS12 Asp89 reaction) describe the same activity at slightly different granularity; they are siblings in the current ontology, not ancestor and descendant, so this row is accepted alongside GO:0103039 and is not TOO_GENERAL. Supporting Evidence: PMID:21169565 RimO (previously shown to be required for this post-translational modification) file:ECOLI/rimO/rimO-uniprot.txt Mutant shows a lack of methylthiolation of protein uS12 and a decrease in transcription of a subset of genes. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro radical SAM mapping to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to GO:0051539: RimO binds two [4Fe-4S] clusters (IDA, PMID:19736993). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:19736993 Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. file:ECOLI/rimO/rimO-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228840 (RimO subfamily). Reason: Core cofactor binding. RimO, like MiaB, binds two [4Fe-4S] clusters: the radical SAM cluster that binds and cleaves SAM and the MTTase-domain auxiliary cluster that activates the exogenous sulfur; both are retained through catalysis (PMID:23542644). Supporting Evidence: PMID:19736993 Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. file:ECOLI/rimO/rimO-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:19736993 Characterization of RimO, a new member of the methylthiotran... | ACCEPT | Summary: Spectroscopy and Fe/S analysis of purified E. coli RimO: one [4Fe-4S] cluster as isolated, two after reconstitution. Reason: Core cofactor binding. RimO, like MiaB, binds two [4Fe-4S] clusters: the radical SAM cluster that binds and cleaves SAM and the MTTase-domain auxiliary cluster that activates the exogenous sulfur; both are retained through catalysis (PMID:23542644). Supporting Evidence: PMID:19736993 Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. file:ECOLI/rimO/rimO-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/InterPro mapping (UR000086055). Reason: Core cofactor binding. RimO, like MiaB, binds two [4Fe-4S] clusters: the radical SAM cluster that binds and cleaves SAM and the MTTase-domain auxiliary cluster that activates the exogenous sulfur; both are retained through catalysis (PMID:23542644). Supporting Evidence: PMID:19736993 Reconstitution of this form of RimO (RimO(rcn)) with (57)Fe and sodium sulfide results in a protein that contains two [4Fe-4S](2+) clusters, similar to MiaB. file:ECOLI/rimO/rimO-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:0103039 protein methylthiotransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/Rhea mapping (RHEA:37087, EC 2.8.4.4): methylthiolation of Asp89 of ribosomal protein uS12 with two SAM. Reason: Core molecular function, and the RHEA-defined reaction term for RimO, parallel to GO:0035597 for MiaB. GO:0103039 is a child of catalytic activity, acting on a protein (GO:0140096), which correctly separates RimO's protein substrate from the tRNA-modifying subfamilies. The activity is established experimentally in E. coli (PMID:18252828, PMID:19736993, PMID:21169565). Supporting Evidence: file:ECOLI/rimO/rimO-uniprot.txt Catalyzes the methylthiolation of the residue Asp-89 of ribosomal protein uS12. PMID:19736993 We also show by mass spectrometry that RimO(rcn) catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. PMID:18252828 Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:19736993 Characterization of RimO, a new member of the methylthiotran... | NEW | Summary: RimO binds S-adenosyl-L-methionine at the unique iron of its radical SAM [4Fe-4S] cluster and reductively cleaves it to 5'-deoxyadenosine during methylthiolation of the uS12 Asp88 peptide mimic. Reason: Not in GOA. Added per the RADICAL_SAM treatment rule and for consistency with miaB, CDK5RAP1 and CDKAL1. This is a molecular function, so the participation test is met directly: purified E. coli RimO itself cleaves SAM to 5'-dA in the presence of its peptide substrate (PMID:19736993). The T. maritima RimO crystal structure shows SAM bound to the radical SAM cluster (PMID:23542644), and UniProt records an exchangeable SAM on that cluster. Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: PMID:19736993 Results from the HPLC-based kinetic assays imply that the reductive cleavage of SAM by RimOrcn to generate 5β²-dA takes place in the presence of the substrate analog P1. file:ECOLI/rimO/rimO-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 One cluster binds S-adenosylmethionine and generates an Adoβ’ radical via a well-established mechanism. |
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Download this section (compressed HTML)Q: GO:0103039 protein methylthiotransferase activity (the RHEA:37087 uS12 Asp89 reaction) and GO:0035599 aspartic acid methylthiotransferase activity (peptidyl-Asp beta-methylthiolation) are siblings under GO:0035596. Should GO:0103039 be is_a GO:0035599, and should GO:0035599 be is_a GO:0140096 catalytic activity, acting on a protein? Should one be merged into the other?
Q: With GO:0018339 (peptidyl-L-beta-methylthioaspartic acid biosynthesis) obsoleted as a pathway variant, what biological process, if any, should RimO carry: a generic protein modification process term, or a ribosome biogenesis/maturation term if modification is shown to occur during subunit assembly?
Q: What does YcaO contribute to uS12 methylthiolation: is it a sulfur-delivery or substrate-presenting partner of RimO, or does it act indirectly?
Experiment: Assay purified RimO on isolated uS12, 30S subunits, assembly intermediates and 70S ribosomes (with and without YcaO) to define the physiological substrate and achieve full turnover in vitro.
Experiment: Compare decoding accuracy, ribosome assembly and streptomycin sensitivity of wild-type, rimO deletion and uS12 D88 substitution strains to find the function of the methylthio-Asp modification.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate state of uS12 for RimO (free protein, assembly intermediate or mature subunit), the role of YcaO, and the functional consequence of Asp88 beta-methylthiolation for the ribosome are unknown.
BIOLOGY BP_DARK
What is known: RimO is the methylthiotransferase for uS12 Asp88; purified RimO modifies an Asp88-loop peptide with SAM cleavage; rimO and ycaO deletions impair the modification in vivo.
Significance: The modification sits at a universally conserved residue near the decoding center, but no phenotype in translation has been tied to it.
What would resolve it: In vitro assays on defined ribosomal substrates and translation-fidelity/assembly phenotyping of rimO and uS12 D88 mutants.
Provenance (the field's own admissions):
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