ABSTRACT
The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to 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, Archaea, and eukaryotes. C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product. The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967. C1407, which is also m5C in natural 16S RNA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition.
Subject(s)
Cytosine/analogs & derivatives , Escherichia coli/enzymology , Methyltransferases/isolation & purification , RNA, Ribosomal, 16S/chemistry , 5-Methylcytosine , Amino Acid Sequence , Cloning, Molecular , Cytosine/chemistry , Cytosine/metabolism , DNA Methylation , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Magnesium/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Open Reading Frames , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Substrate SpecificityABSTRACT
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.
