| Item | Key finding | Evidence type/method | Key quantitative/statistical detail (if any) | Primary source with DOI/URL and year | Citation ID |
|---|---|---|---|---|---|
| Identity | **rlmC** in *Escherichia coli* K-12 corresponds to **YbjF/RumB**, the dedicated **23S rRNA (uracil-747)-C5 methyltransferase** RlmC, distinct from RlmD (U1939) and TrmA (tRNA U54). | Gene-function assignment from knockout-based modification mapping; comparative family/evolution analyses | E. coli has **three** related COG2265 m5U methyltransferases with distinct targets | Madsen et al., *Nucleic Acids Research* (2003), DOI: 10.1093/nar/gkg657, https://doi.org/10.1093/nar/gkg657; Desmolaize et al., *Nucleic Acids Research* (2011), DOI: 10.1093/nar/gkr626, https://doi.org/10.1093/nar/gkr626 | (pqac-00000002, pqac-00000005) |
| Reaction | RlmC catalyzes **SAM/AdoMet-dependent C5 methylation of uridine 747** in 23S rRNA, generating **m5U747** (ribothymidine). | In vivo loss-of-modification mapping by MALDI-MS in knockout strains; family/mechanistic inference for SAM-dependent m5U MTases | Loss of a single methyl group gives a **~14 Da** mass decrease in the relevant 23S rRNA fragment | Madsen et al. (2003), DOI: 10.1093/nar/gkg657, https://doi.org/10.1093/nar/gkg657; Auxilien et al., *RNA* (2011), DOI: 10.1261/rna.2323411, https://doi.org/10.1261/rna.2323411 | (pqac-00000000, pqac-00000006) |
| Substrate | The mapped target is the **23S rRNA segment containing U747**, specifically localized to the **U746-U747-G748** region; evidence supports methylation at **U747**, not neighboring residues. | RNase T1 digestion plus MALDI-MS of defined oligonucleotides from WT vs knockout rRNA | Decamer mass shift **3253.4 → 3239.6**; in an rrmA-deficient background a heptamer at **m/z 2268.3** enabled localization to the trinucleotide region | Madsen et al. (2003), DOI: 10.1093/nar/gkg657, https://doi.org/10.1093/nar/gkg657 | (pqac-00000000) |
| Location | The enzyme functions in the **cytoplasm** on **23S rRNA/large ribosomal subunit biogenesis substrates**; the modified nucleotide lies in **hairpin 35** of 23S rRNA and projects toward the **nascent peptide exit tunnel**. | Structural/functional interpretation from ribosome mapping and comparative review | No direct subcellular fractionation reported for RlmC in the cited evidence | Auxilien et al. (2011), DOI: 10.1261/rna.2323411, https://doi.org/10.1261/rna.2323411; Ofengand & Del Campo, *EcoSal Plus* (2004), DOI: 10.1128/ecosalplus.4.6.1, https://doi.org/10.1128/ecosalplus.4.6.1 | (pqac-00000006, pqac-00000001) |
| Biological role | RlmC is part of the **rRNA modification pathway** supporting maturation and functional tuning of the 50S subunit; available evidence suggests the true substrate may be an **assembly intermediate/RNP or intact 50S particle**, rather than naked RNA. | Negative in vitro reconstitution results with recombinant protein and transcript substrates; review-based functional interpretation | Recombinant/purified RlmC showed **no detectable activity** on isolated 23S rRNA from the mutant or on a **694-767 nt** transcript in reported assays | Ofengand & Del Campo (2004), DOI: 10.1128/ecosalplus.4.6.1, https://doi.org/10.1128/ecosalplus.4.6.1 | (pqac-00000001) |
| Phenotypes | **rlmC deletion** causes a **mild phenotype** overall but is associated with **17S rRNA precursor accumulation**, especially at **20°C**, indicating a subtle role in small-subunit rRNA processing/overall ribosome homeostasis; no major ribosomal assembly intermediate accumulation like **rlmE**. | Keio knockout phenotyping; RT-qPCR for 17S precursor; sucrose gradient centrifugation under dissociating/associating Mg2+ conditions; reporter-expression assays | 17S accumulation detected at **20°C not 37°C**; gradients examined at **1 mM** and **10 mM Mg2+**; some rRNA MT knockouts showed up to **10-fold** RFP reduction in reporter assays, with rlmC included among strains with reduced expression burden tolerance | Pletnev et al., *Frontiers in Genetics* (2020), DOI: 10.3389/fgene.2020.00097, https://doi.org/10.3389/fgene.2020.00097 | (pqac-00000007, pqac-00000008, pqac-00000010) |
| Recent developments | Recent work emphasizes **high-resolution ribosome structure/modification mapping** and broader bacterial **epitranscriptomics/modomics** as the main route for contextualizing m5U747; direct 2023-2024 E. coli-specific RlmC studies are limited, so current understanding still relies heavily on foundational mapping and comparative analyses. | Recent structural/modification-mapping literature and synthesis with older primary assignment papers | 2024 studies highlight species-specific confirmation of many rRNA modifications but do not substantially revise the core E. coli RlmC assignment | González-López et al., *Scientific Reports* (2024), DOI: 10.1038/s41598-024-64868-x, https://doi.org/10.1038/s41598-024-64868-x; Pletnev et al. (2020), DOI: 10.3389/fgene.2020.00097, https://doi.org/10.3389/fgene.2020.00097 | (pqac-00000008) |
| Applications | RlmC serves as a **reference enzyme/site** for **rRNA modification mapping**, **comparative evolution of m5U methyltransferases**, and potential **ribosome-targeted antimicrobial research**, especially in studies comparing single-specificity enzymes (RlmC/RlmD) with dual-specificity homologs (RlmCD). | MALDI-MS mapping workflows; comparative enzymology and structure-guided analyses | The classic assignment relied on diagnostic **single-methyl (~14 Da)** mass shifts in specific oligoribonucleotides | Madsen et al. (2003), DOI: 10.1093/nar/gkg657, https://doi.org/10.1093/nar/gkg657; Jiang et al., *PLOS Pathogens* (2018), DOI: 10.1371/journal.ppat.1007379, https://doi.org/10.1371/journal.ppat.1007379 | (pqac-00000000, pqac-00000004) |


*Table: This table summarizes the main evidence supporting the functional annotation of E. coli K-12 RlmC (UniProt P75817), including identity, catalytic activity, substrate assignment, phenotypes, and current research uses. It is useful as a compact evidence map linking each claim to the underlying method and source.*