RlmC (formerly RumB/YbjF) is a 23S rRNA (uracil(747)-C(5))-methyltransferase (EC 2.1.1.189) in E. coli K12 that catalyzes the formation of 5-methyluridine at position 747 (m5U747) in 23S rRNA using S-adenosyl-L-methionine as methyl donor. It belongs to the COG2265 cluster of RNA m5U methyltransferases, which in E. coli includes three paralogs with distinct specificities: TrmA (tRNA U54), RlmC (23S rRNA U747), and RlmD/RumA (23S rRNA U1939). Function was established in vivo by Madsen et al. (2003, PMID:12907714) using a ybjF knockout strain, which specifically lacked only the m5U747 modification. RlmC contains an N-terminal [4Fe-4S] cluster (predicted by HAMAP based on the conserved CX5CGGC motif established in the paralog RumA/RlmD) and a C-terminal SAM-dependent methyltransferase fold with a catalytic cysteine (Cys334) that forms a covalent Michael adduct with the target uracil during catalysis. In some Gram-positive bacteria such as B. subtilis, a single enzyme (RlmCD) performs both the U747 and U1939 modifications, suggesting evolutionary specialization of the COG2265 paralogs in E. coli.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0070041 rRNA (uridine-C5-)-methyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for rRNA (uridine-C5-)-methyltransferase activity based on phylogenetic inference (PANTHER). This is the most specific molecular function term for RlmC and is strongly supported by the in vivo knockout evidence from Madsen et al. 2003 (PMID:12907714), which showed loss of m5U747 modification specifically in the ybjF/rlmC deletion strain. Reason: This is the correct and most specific molecular function annotation for RlmC. The IBA inference from PANTHER is well supported by experimental IMP evidence (PMID:12907714) demonstrating that RlmC specifically methylates U747 at C5 in 23S rRNA. Supporting Evidence: PMID:12907714 Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification file:ECOLI/rlmC/rlmC-deep-research-bioreason-sft.md BioReason domain architecture analysis confirms RlmC family assignment (IPR011825) and the TrmA-like catalytic mechanism, consistent with rRNA (uridine-C5-)-methyltransferase activity file:ECOLI/rlmC/rlmC-deep-research-falcon.md These data directly support that **RlmC is required in vivo for m5U747 formation**. |
| GO:0070475 rRNA base methylation | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for rRNA base methylation based on phylogenetic inference (PANTHER). Correct biological process for RlmC. The m5U747 modification is a base methylation on 23S rRNA. Reason: Accurately describes the biological process: RlmC catalyzes methylation of a base (uracil C5) in rRNA. Supported by in vivo evidence from PMID:12907714. Supporting Evidence: PMID:12907714 Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification file:ECOLI/rlmC/rlmC-deep-research-falcon.md This assignment explicitly distinguishes RlmC from the related paralogs **TrmA** (tRNA U54 methyltransferase) and **RlmD/RumA** (23S rRNA U1939 methyltransferase). |
| GO:0005506 iron ion binding | IEA GO_REF:0000104 | MODIFY | Summary: IEA annotation for iron ion binding based on UniRule (UR000100395). RlmC contains a predicted [4Fe-4S] cluster coordinated by Cys3, Cys11, Cys14, and Cys87 (HAMAP-Rule:MF_01012). The [4Fe-4S] cluster has been experimentally demonstrated in the paralog RumA/RlmD (PMID:15181002, PMID:15016356) and the cysteine motif CX5CGGC is conserved in RlmC. While iron ion binding is technically correct, a more precise term would be 4 iron, 4 sulfur cluster binding (GO:0051539), which is already annotated in the UniProt DR lines. Reason: Iron ion binding is too general. The iron is bound as part of a [4Fe-4S] cluster, not as free iron ions. GO:0051539 (4 iron, 4 sulfur cluster binding) is a more precise and informative annotation. The [4Fe-4S] cluster is well-established in this enzyme family (PMID:15181002). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:15181002 Sequence data base searches revealed that RumA homologs are widespread in various kingdoms of life and contain a conserved and unique iron-sulfur cluster binding motif, CX(5)CGGC |
| GO:0006396 RNA processing | IEA GO_REF:0000120 | MODIFY | Summary: IEA annotation for RNA processing based on combined automated methods (InterPro:IPR010280). RNA processing is overly broad for what RlmC does. RlmC performs a specific chemical modification (methylation) of rRNA, not processing in the sense of cleavage, splicing, or end-maturation. The more appropriate term is rRNA methylation (GO:0031167) or rRNA base methylation (GO:0070475), both of which are already annotated. Reason: RNA processing is too general and somewhat misleading for a methyltransferase. Methylation is a modification, not processing. The correct biological process terms (rRNA methylation, rRNA base methylation) are already present. Proposed replacements: rRNA methylation Supporting Evidence: PMID:12907714 here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry file:ECOLI/rlmC/rlmC-deep-research-falcon.md loss of m5U747 produces **subtle/conditional defects** in rRNA maturation/homeostasis rather than catastrophic ribosome assembly failure. |
| GO:0008173 RNA methyltransferase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: IEA annotation for RNA methyltransferase activity. This is correct but less specific than GO:0070041 (rRNA (uridine-C5-)-methyltransferase activity) and GO:0016436 (rRNA (uridine) methyltransferase activity), both of which are already annotated. This is a valid parent term but redundant when the more specific child terms are present. Reason: Correct but redundant with the more specific terms GO:0070041 and GO:0016436 that are already annotated. Keeping as non-core since it adds no information beyond what the more specific terms provide. |
| GO:0008757 S-adenosylmethionine-dependent methyltransferase activity | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: IEA annotation for SAM-dependent methyltransferase activity based on ARBA machine learning. Correct: RlmC uses S-adenosyl-L-methionine as methyl donor. The catalytic reaction is well-defined (Rhea:RHEA:42628). However, this is a general parent term and less specific than GO:0070041. Reason: Correct cofactor usage but a general parent of the more specific rRNA methyltransferase terms already present. The SAM-dependent mechanism is confirmed by the UniProt catalytic activity annotation and the family membership in COG2265 SAM-dependent RNA m5U methyltransferases. Supporting Evidence: PMID:12907714 Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA file:ECOLI/rlmC/rlmC-deep-research-falcon.md RlmC is a **23S rRNA (uracil(747))-C(5)-methyltransferase** (EC **2.1.1.189**) that catalyzes: |
| GO:0009451 RNA modification | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: IEA annotation for RNA modification based on ARBA. Correct but very general. More specific child terms (rRNA base methylation GO:0070475, rRNA methylation GO:0031167) are already annotated. Reason: Correct but very general parent of more specific biological process terms already annotated. Redundant with GO:0070475 and GO:0031167. |
| GO:0016070 RNA metabolic process | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: IEA annotation for RNA metabolic process based on InterPro (IPR011825). Extremely broad term. While technically true, this is uninformative given the much more specific annotations already present. Reason: Extremely broad parent term. Redundant with more specific biological process annotations (rRNA base methylation, rRNA methylation). Provides no additional functional insight. |
| GO:0016436 rRNA (uridine) methyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: IEA annotation for rRNA (uridine) methyltransferase activity based on InterPro (IPR011825). This is a correct molecular function annotation at an intermediate specificity level. GO:0070041 (rRNA (uridine-C5-)-methyltransferase activity) is more specific and also annotated. Since the evidence from PMID:12907714 specifically demonstrates C5 methylation, the more specific term is preferred. Reason: The evidence supports the more specific term GO:0070041 (rRNA (uridine-C5-)- methyltransferase activity), which specifies the C5 position. Consistent with the action for the IMP-evidenced annotation of the same term. Proposed replacements: rRNA (uridine-C5-)-methyltransferase activity |
| GO:0031167 rRNA methylation | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: IEA annotation for rRNA methylation based on UniRule (UR000100395). This is an appropriate biological process term for RlmC. It is a parent of GO:0070475 (rRNA base methylation), which is already annotated with experimental (IMP) evidence. Keeping as non-core since the more specific child term is present. Reason: Correct but parent of the more specific GO:0070475 (rRNA base methylation) which is annotated with IMP evidence. |
| GO:0070041 rRNA (uridine-C5-)-methyltransferase activity | IEA GO_REF:0000104 | ACCEPT | Summary: IEA annotation for rRNA (uridine-C5-)-methyltransferase activity based on UniRule (UR000100395). Duplicate of the IBA annotation with the same GO term from GO_REF:0000033. Both are correct; this IEA provides independent computational support. Reason: Correct and most specific molecular function term. Independent computational support for the IBA annotation from PANTHER. Supporting Evidence: PMID:12907714 Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification |
| GO:0070475 rRNA base methylation | IMP PMID:12907714 Identifying the methyltransferases for m(5)U747 and m(5)U193... | ACCEPT | Summary: IMP annotation for rRNA base methylation from Madsen et al. 2003. This is the key experimental evidence for RlmC function, based on comparison of methylation patterns between wild-type and ybjF knockout strains using MALDI mass spectrometry. The knockout specifically lost m5U747 methylation. Reason: Core experimental evidence (IMP) demonstrating that RlmC is required for rRNA base methylation in vivo. The MALDI-MS analysis directly showed loss of m5U747 in the knockout strain. This is the most informative biological process annotation with direct experimental support. Supporting Evidence: PMID:12907714 Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification |
| GO:0016436 rRNA (uridine) methyltransferase activity | IMP PMID:12907714 Identifying the methyltransferases for m(5)U747 and m(5)U193... | MODIFY | Summary: IMP annotation for rRNA (uridine) methyltransferase activity from Madsen et al. 2003. Based on the same knockout experiment that demonstrated loss of m5U747 in the ybjF- strain. While IMP (inferred from mutant phenotype) is technically the correct evidence code for this knockout study, a more specific MF term GO:0070041 would be preferred. Reason: The evidence from PMID:12907714 supports the more specific term GO:0070041 (rRNA (uridine-C5-)-methyltransferase activity) since the study specifically demonstrated C5 methylation of uridine. Upgrading to the more specific term. Proposed replacements: rRNA (uridine-C5-)-methyltransferase activity Supporting Evidence: PMID:12907714 There are three sites of m(5)U modification in Escherichia coli stable RNAs |
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Download this section (compressed HTML)Q: What is the functional significance of the m5U747 modification for ribosome assembly or translational fidelity? The original knockout study (PMID:12907714) did not report growth phenotypes.
Q: Has the [4Fe-4S] cluster in RlmC been experimentally verified, or is this inference based solely on sequence conservation with RumA/RlmD?
Q: Why could recombinant RlmC not be obtained in active form in vitro (PMID:12907714)? Is this related to [4Fe-4S] cluster assembly or protein folding requirements?
Experiment: Phenotypic characterization of an rlmC deletion strain under stress conditions (e.g., sublethal antibiotic concentrations, temperature stress, competitive growth). The original study (PMID:12907714) focused on biochemical identification of the modification rather than phenotypic consequences.
Hypothesis: Loss of m5U747 may confer subtle growth disadvantages under stress conditions or when combined with loss of other rRNA modifications.
Experiment: Spectroscopic characterization (UV-vis, EPR) of purified RlmC to directly demonstrate the presence of a [4Fe-4S] cluster, analogous to the studies performed on RumA/RlmD (PMID:15181002).
Hypothesis: RlmC contains a [4Fe-4S] cluster coordinated by Cys3, Cys11, Cys14, and Cys87, analogous to the cluster in RumA/RlmD.
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