Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE.
The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.
Basturea
Georgeta N
GN
Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL 33101-6129, USA.
Deutscher
Murray P
MP
D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases; C525730:RsmE protein, E coli