Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.
RsmF methylates C1407 in the stem of helix 44 near the 3’-end of 16S rRNA within assembled 30S subunits (it does not work on naked 16S rRNA or within tight-couple 70S ribosome). The ribosomal protein S12 is specially important. The enzyme has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m5C RNA MTases (including RsmB). However, E. coli RsmF has also a C-terminal PUA-domain similar to that in pseudouridine synthases (Pus) and archaeosine-specific transglycosylases (Tgt). It also contains extended regions of positive electrostatic potential that differ from other RNA MTase structures. Methylation of C1407 by RsmF is impeded by Sgm methyltransferase that catalyzes the formation of m7G on its adjacent G1405 target on the 30S ribosomal subunit. Thus resistance to certain aminoglycosides depends on the interplay of two different methyltransferases: RsmF and Sgm. RsmF is conserved in bacteria.