Purification, cloning, and characterization of the 16S RNA m(5)C967 methyltransferase from Escherichia coli

The methyltransferase that forms m(5)C967 in Escherichia colt small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was i dentified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed tb be distinct due to a DNA sequencing error, The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, A rchaea, and eukaryotes. C-Terminal sequencing of the overexpressed and: aff inity-purified protein by mass spectrometry methods verified the sequence e xpected for the gene product. The recombinant protein exhibited the same sp ecificity as the previously described native enzyme; that is, it formed onl y m(5)C and only at position 967. C1407, which is also m5C in natural 16S R NA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for a dded magnesium, suggesting that extensive secondary or tertiary structure i n the RNA substrate may not be a requirement for recognition.