RlmI specifically methylates the C5-residue of conserved cytosine 1962 (m5C), located in position just precedding hairpin 71 of domain IV in 23S rRNA. In vitro, it works on naked 23S rRNA isolated from the yccW knockout strain, and not in assembled 50S subunits or ribosomes. The methyl donor group is AdoMet. The RlmI molecule comprises three domains: the N-terminal PUA domain; the central domain, which resembles a domain previously found in RNA:5-methyluridine MTases, and the C-terminal catalytic domain, which contains the AdoMet-binding site. The central and C-terminal domains are linked by a beta-hairpin structure that has previously been observed in several MTases acting on nucleic acids or proteins. On evolutionary point of view, RlmI is similar to known m5U (5-methyluridine) RNA methyltransferases.