rlmC (RumB/YbjF) - Research Notes

Gene Identity

• UniProt (reviewed): P75817 (RLMC_ECOLI, E. coli K12/MG1655)
• UniProt (unreviewed): B1X800 (E. coli K12/DH10B) -- same protein, different strain
• Synonyms: rumB, ybjF
• EC: 2.1.1.189
• Gene: b0859 / JW0843
• Full name: 23S rRNA (uracil(747)-C(5))-methyltransferase RlmC

Core Function

RlmC catalyzes the formation of 5-methyluridine at position 747 (m5U747) in 23S rRNA, using S-adenosyl-L-methionine (SAM/AdoMet) as the methyl donor.

Key Literature

PMID:12907714 - Madsen et al. 2003, Nucleic Acids Res 31:4738-4746

"Identifying the methyltransferases for m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry."

This is the definitive paper establishing the function of RlmC (then called YbjF). Key findings:
- Three m5U sites in E. coli stable RNAs: U54 in tRNA (TrmA), U747 in 23S rRNA (YbjF/RumB/RlmC), and U1939 in 23S rRNA (YgcA/RumA/RlmD)
- YbjF function was defined in vivo by engineering a ybjF knockout strain PMID:12907714
- YbjF(-) strains showed ONLY the loss of m5U747 modification PMID:12907714
- Could not get recombinant YbjF to retain in vitro activity PMID:12907714
- Proposed the name RumB (RNA uridine methyltransferase B)

PMID:21824914 - Desmolaize et al. 2011, Nucleic Acids Res 39:9368-75

"A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA."

Key comparative finding:
- In E. coli: three separate COG2265 paralogs (TrmA, RlmC, RlmD) for three m5U sites
- In B. subtilis: a single enzyme YefA/RlmCD handles both m5U747 and m5U1939
- PMID:21824914
- PMID:21824914
- Suggests evolutionary specialization of COG2265 paralogs

PMID:21051506 - Auxilien et al. 2011, RNA 17:45-53

"Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases."

Key evolutionary findings:
- PMID:21051506
- RlmC (formerly RumB) specifically modifies m5U747 in 23S rRNA
- In Pyrococcus abyssi, PAB0760 has RlmC-like activity despite being more closely related to RlmD in sequence

PMID:30388185 - Jiang et al. 2018, PLoS Pathog 14:e1007379

"Unveiling the structural features that determine the dual methyltransferase activities of Streptococcus pneumoniae RlmCD."

• Structure of S. pneumoniae RlmCD in complex with RNA substrate containing U747 or U1939
• PMID:30388185
• F145 side-chain rearrangement discriminates between U747- and U1939-containing substrates

PMID:15016356 - Lee et al. 2004, Structure 12:397-407

"Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase."

• Structure of the paralog RumA (RlmD) -- relevant by homology to RlmC
• Three domains: N-terminal TRAM domain, central [4Fe-4S] cluster domain, C-terminal SAM-dependent MTase domain
• The [4Fe-4S] cluster is coordinated by four conserved cysteines in a CX5CGGC motif

PMID:15181002 - Agarwalla et al. 2004, J Biol Chem 279:34123-9

"Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA."

• The [4Fe-4S] cluster in RumA/RlmD is redox-active but the methyltransferase reaction does not involve a redox step
• PMID:15181002
• Oxidation of the cluster leads to decomposition -- potential regulatory mechanism under oxidative stress

PMID:12003490 - Agarwalla et al. 2002, J Biol Chem 277:8835-8840

Initial characterization of RumA, establishing the presence of a [4Fe-4S] cluster in this family.

PMID:10369764 - Gregory & Dahlberg 1999, J Mol Biol 289:827-34

"Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA."

• L22 mutation influences modification at m5U747 position
• PMID:10369764
• This indicates that m5U747 is in domain II of 23S rRNA and its accessibility is influenced by ribosomal protein L22

PMID:17459887 - Urbonavicius et al. 2007, Nucleic Acids Res 35:3297-305

"Amino acid residues of the E. coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity."

• Establishes conserved residues across COG2265 family (TrmA, RumA/RlmD, RumB/RlmC)
• PMID:17459887
• Supports modular evolution model: conserved catalytic domain + different RNA-binding domains

Domain Architecture (from UniProt and InterPro)

• N-terminal region: [4Fe-4S] cluster binding (Cys3, Cys11, Cys14, Cys87) -- HAMAP prediction
• Central/C-terminal: SAM-dependent methyltransferase fold (Rossmann-like)
• Active site: Cys334 (nucleophile for covalent Michael adduct at uracil C6)
• SAM binding: residues 212, 241, 262, 307

Catalytic Mechanism

The TrmA/RlmC/RlmD family uses a common mechanism:
1. Nucleophilic Cys attacks C6 of the target uracil, forming a covalent Michael adduct
2. This activates C5 for electrophilic methyl transfer from SAM
3. Beta-elimination releases the methylated product (m5U) and regenerates the Cys

The [4Fe-4S] Cluster Question

UniProt lists RlmC as containing a [4Fe-4S] cluster based on HAMAP rule MF_01012. The binding motif CX5CGGC is present in the N-terminal region (Cys3, Cys11, Cys14, Cys87). This is well-established for the paralog RumA/RlmD (PMID:12003490, PMID:15016356, PMID:15181002) but has not been directly demonstrated experimentally for RlmC. The annotation is based on sequence conservation and is likely correct given the high conservation of the cysteine motif. The role of the [4Fe-4S] cluster is structural rather than catalytic -- the methyltransferase reaction does not involve a redox step (PMID:15181002).

Biological Significance

• m5U747 is in domain II of 23S rRNA, near the peptidyl transferase center region
• The modification is phylogenetically conserved across bacteria
• In B. subtilis, both m5U747 and m5U1939 are present (catalyzed by a single enzyme RlmCD)
• Loss of m5U747 alone (ybjF/rlmC knockout) was demonstrated without reported growth defects in the original Madsen et al. 2003 study, though detailed phenotypic analysis was not the focus
• The modification likely contributes to local rRNA structural stability and ribosome function