RlmKL is a bifunctional protein (700 amino acids) that catalyzes the in vitro formation of two different methylated residues (m7G and m2G) within the same helix 74 in domain IV (Peptidyl Transferase Center) of 23S rRNA purified from the ycbY knock-out strain (positions 2069 and 2445 respectively). Assembled 50 S subunits are not a substrate for the RlmKL. It contains two Rossmann-fold methyltransferase domains. The N-terminal domain (RlmL domain) is a member of COG0116 and consists of a MTase domain and a RNA-binding THUMP domain. The C-terminal domain (RlmK domain) is a MTase and member of COG1092. AdoMet is the methyl group donor. RlmKL was shown to have an activity to unwind 23S rRNA helix 74 during substrate recognition and methylation. The proposed mechanism for the methylation of two target sites: first the 2069 m7G is introduced by RlmK domain, then the 2445 m2G by RlmL domain. The presence of RlmK domain facilitates methylation in position 2445 but the reactions are independent. P-loop of helix 80 is critical for substrate recognition. Phylogenetic analysis suggests that bifunctional RlmKL is only present in Gammaproteobacteria, and that the separated RlmK and RlmL are found in the genus Neisseria.