title: Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli.
abstract: An Escherichia coli open reading frame, ygcA, was identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762). We have cloned, expressed, and purified the 50-kDa protein encoded by ygcA. The purified enzyme catalyzed the AdoMet-dependent methylation of 23 S rRNA but did not act upon 16 S rRNA or tRNA. A high performance liquid chromatography-based nucleoside analysis identified the reaction product as 5-methyluridine. The enzyme specifically methylated U1939 as determined by a nuclease protection assay and by methylation assays using site-specific mutants of 23 S rRNA. A 40-nucleotide 23 S rRNA fragment (nucleotide 1930--1969) also served as an efficient substrate for the enzyme. The apparent K(m) values for the 40-mer RNA oligonucleotide and AdoMet were 3 and 26 microm, respectively, and the apparent k(cat) was 0.06 s(-1). The enzyme contains two equivalents of iron/monomer and has a sequence motif similar to a motif found in iron-sulfur proteins. We propose to name this gene rumA and accordingly name the protein product as RumA for RNA uridine methyltransferase.
authors.affiliation: Department of Biochemistry, University of California at San Francisco, San Francisco, California 94143-0448, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Agarwalla
authors.forename: James T
authors.initials: JT
authors.lastname: Kealey
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Santi
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D008745:Methylation; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012338:RNA, Ribosomal, 23S; C093591:RumA protein, Bacteria; C009182:ribothymidine; D012359:tRNA Methyltransferases; D014529:Uridine