Purification, cloning, and characterization of the 16 S RNA m2G1207 methyltransferase from Escherichia coli.
The methyltransferase that forms m2G1207 in Escherichia coli small subunit rRNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme as the open reading frame yjjT (SWISS-PROT accession number ). The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA. By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the enzyme, it was shown that of the three naturally occurring m2G residues, only m2G1207 was formed. Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mM Mg2+, both 2 mM EDTA and 6 mM Mg2+ markedly reduced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.
Tscherne
J S
JS
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Nurse
K
K
Popienick
P
P
Ofengand
J
J
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008274:Magnesium / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases; C116918:rsmC protein, E coli; D008274:Magnesium