RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.