ygiQ was in the y-ome, with membership of the radical SAM superfamily as its only annotation, and was co-expressed with well-annotated translation genes; this co-expression is consistent with the rRNA-modifying function later established for the gene.

A screen of 15 uncharacterised and 6 known E. coli RNA methyltransferases under anaerobic conditions identified ygiQ as the gene required for the 5'(S)-methylations of D2449 and Cm2498 of 23S rRNA; plasmid-borne ygiQ restored both modifications, and the authors rename ygiQ as rlmX.

RlmX is a cobalamin-dependent radical SAM methyltransferase built from a cobalamin-binding domain, a radical SAM domain and a DUF3362 domain, with cobalamin-binding, SAM-binding and [4Fe-4S]-binding motifs identified by comparison of an AlphaFold2 model with crystal structures of cobalamin-dependent radical SAM methyltransferases.

Reconstituted recombinant RlmX contains a single [4Fe-4S] cluster: iron quantification gave 3.69 iron atoms per protein, about 92% occupancy of one cluster, and methylcobalamin binding was observed by its 320 nm absorbance.

Cys386, Cys390 and Cys393 are the [4Fe-4S] cluster ligands and are each individually essential; G160N in the cobalamin-binding GG motif abolishes methylation; and Glu525, which contacts the SAM ribose, is required for full activity.

The RlmX-specific cysteine triad Cys453, Cys458 and Cys464 lies on the protein surface and is thought to contribute to RNA recognition through zinc binding; all three residues are essential for methylation. It is not a second iron-sulfur cluster.

Holo-RlmX methylates deproteinized 23S rRNA and in vitro transcribed 23S rRNA, with domain V as the minimum efficient substrate, but does not methylate the assembled 50S subunit, so the modifications are installed early in subunit assembly.

Methylation is SAM-dependent and the methyl group derives from SAM, shown by transfer of CD3 from CD3-SAM; both the [4Fe-4S] cluster and methylcobalamin are required for an active holoenzyme, with methylcobalamin acting as the immediate methyl donor and a second SAM regenerating it.

The modifications require ferric iron and cobalamin in the growth medium, and both modifications plus RlhA-dependent ho5C2501 rise under anaerobic growth, with RlmX additionally more active anaerobically despite unchanged rlmX mRNA.

A double knockout lacking rlmX and rlhA grows slowly anaerobically, rescued by wild-type rlmX but not by the catalytically inactive G160N or C390A, and its ribosomes transfer peptide at 64% of the wild-type rate, while 70S initiation and P-site tRNA binding are unaffected.

UniProt records one [4Fe-4S] cluster coordinated by three cysteines and an exchangeable S-adenosyl-L-methionine, consistent with the measured cluster content, but still lists no gene name rlmX and no function.

The report concludes that YgiQ is RlmX, the cobalamin-dependent radical SAM methyltransferase that methylates the ribose C5'(S) of two 23S rRNA nucleotides in the peptidyl-transferase centre. This central conclusion is confirmed by the full text of PMID:41380683.