Purification, Cloning, and Characterization of the 16 S RNA m2G1207 Methyltransferase from Escherichia coli

Abstract

The methyltransferase that forms m 2 G1207 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 P39406). 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 m 2 G residues, only m 2 G1207 was formed. Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mM Mg 2؉ , both 2 mM EDTA and 6 mM Mg 2؉ 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.

The FtsJ/RrmJ Heat Shock Protein of Escherichia coli Is a 23 S Ribosomal RNA Methyltransferase

“…This property is shared by RsmA and RsmC enzymes (9,32), and is true of most rRNAmodifying enzymes, with the exception of RsmB (2). RrmJmediated methylation requires magnesium; this requirement has also been reported for activity of other rRNA methyltransferases, and appears to be characteristic of reactions involving ribosomes or ribosomal particles (9).…”

“…Several rRNA methyltransferases, such as RsmB (2), function equally well in EDTA as in the presence of magnesium ions whereas others, like RsmC, require magnesium ions for an efficient methylation reaction (9). The RrmJ methyltransferase shows a stringent dependence on magnesium concentration, with a peak centered around 2 mM magnesium, and its activity is severalfold lower in the presence of 10 mM EDTA or in the presence of 10 mM magnesium (data not shown).…”

“…The nucleotide methylation of rRNAs can modify the susceptibility of ribosomes to antibiotics that target them (6); it is reasonable to expect that the binding of other ligands can be affected as well. In E. coli, three 16 S RNA methyltransferases have been identified, RsmA (also known as KsgA) (7), RsmB (2,8) and RsmC (9). In contrast, little is known about genes involved in 23 S rRNA modifications.…”

Structural and Functional Characterization of Rv2966c Protein Reveals an RsmD-like Methyltransferase from Mycobacterium tuberculosis and the Role of Its N-terminal Domain in Target Recognition

“…RsmC and RsmE that modify m 2 G1207 and m 3 U1498 of 16 S rRNA, respectively (30,31), lack in vitro methyltransferase activity in the absence of ribosomal subunits. To monitor the in vitro activity of Rv2966c, Rv2966c was incubated with 30 S ribosomal subunits in the presence of SAM, and the methyltransferase activity was monitored colorimetrically.…”

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N2-methylguanosine at position 6 in tRNA

“…In Escherichia coli, three m 2 G residues are found in the 16S rRNA (at positions 966, 1207, and 1516) and two in the 23S rRNA (at positions 1835 and 2445) (Andersen and Douthwaite 2006). Genes encoding four out of the five rRNA (m 2 G) methyltransferases (MTases) have been identified in the E. coli chromosome (Tscherne et al 1999;Lesnyak et al 2006Lesnyak et al , 2007Sergiev et al 2006). For the RsmC and RsmD enzymes, crystal structures have been solved (Lesnyak et al 2007;Sunita et al 2007).…”