In the

previous study (Wachino et al., 2006), we purified C-terminal histidine-tagged RmtC with the combination of a pET29a vector and an E. coli BL21(DE3)pLysS. However, only approximately 1 mg of protein was purified from a 1 L culture. Therefore, we generated another expression construct consisting of a pCold-II vector and an E. coli BL21(DE3)pLysS to improve the protein purification productivity. Optimized conditions yielded 8 mg of purified protein per 1 L culture, and its purity seemed very high on SDS-PAGE (data not shown). The native molecular weight (M) of the His6-RmtC protein was determined to be approximately 34 000 by gel filtration chromatography. Purified His6-RmtC protein specifically had an MTase activity against DNA Damage inhibitor an assembled 30S SB203580 mouse ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but no methylation activity was detected against protein-free 16S rRNA in the presence of the methyl group donor S-adenosyl-l-methionine (Fig. 1). This substrate specificity of RmtC to the 30S ribosomal subunit, not to naked 16S rRNA, has been commonly observed among 16S rRNA MTases such as ArmA (G1405), RmtB (G1405), Sgm (G1405), RsmF[YebU] (C1407), and NpmA (A1408), which methylate the nucleotide within the ribosomal A-site (Andersen & Douthwaite, 2006; Liou et al., 2006; Wachino et al., 2007; Savic et al., 2008; Cubrilo et al., 2009; Schmitt et al., 2009). The specific activity of these

16S rRNA MTases for the 30S ribosomal subunit would indicate

that ribosomal proteins play a crucial role in the precise substrate recognition. To determine the precise nucleotide position modified by RmtC, we used three approaches: RNase protection assay, primer extension, and HPLC. [3H]-methyl-labeled 16S rRNA modified by RmtC in vitro was hybridized with oligonucleotides complementary to the rRNA region, and treated with RNaseA, Miconazole and the radioactivity was measured to confirm the region to which the [3H]-methyl group was added. The hybridization of oligonucleotides spanning from G1392 position to the G1421 position was able to maintain the radioactivity after RNaseA treatment (Fig. 2), suggesting that the nucleotide residue methylated by RmtC was located between G1392 and G1421 of 16S rRNA. A primer extension analysis was performed as the second assay for the determination of the methylated position. The 16S rRNA prepared from the 30S ribosomal subunit of E. coli JM109 expressing RmtC was treated with NaBH4/aniline before the primer extension reaction, and the site of methylation was determined by acrylamide gel electrophoresis. By primer extension analysis, one reverse transcription stop was observed at position U1406 as a result of β-elimination at the 3′-end of the N7-position of the nucleotide G1405 (Fig. 3). The termination of transcription at U1406 was not observed without the treatment with NaBH4/aniline (Fig. 3).