The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA

Abstract

A2503 in 23S rRNA of the Gram-negative bacterium Escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. In E. coli, and likely in other species, this adenosine residue is posttranscriptionally modified to m 2 A. The enzyme responsible for this modification was previously unknown. We identified E. coli protein YfgB, which belongs to the radical SAM enzyme superfamily, as the methyltransferase that modifies A2503 of 23S rRNA to m 2 A. Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA as revealed by primer extension analysis and thin layer chromatography. The A2503 modification was restored when YfgB protein was expressed in the yfgB knockout strain. A similar protein was shown to catalyze post-transcriptional modification of A2503 in 23S rRNA in Gram-positive Staphylococcus aureus. The yfgB knockout strain loses in competition with wild type in a co-growth experiment, indicating functional importance of A2503 modification. The location of A2503 in the exit tunnel suggests its possible involvement in interaction with the nascent peptide and raises the possibility that its post-transcriptional modification may influence such an interaction.

YbeA is the m3Ψ methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA

“…Growth competition experiments were performed in duplicate as previously described (Gutgsell et al 2000;Toh et al 2008) starting with approximately equal numbers of xylA-and ybeAknockout cells at the same growth phase or, alternatively, starting under the same conditions with wild-type and ybeA-knockout cells. The xylA gene in the growth comparator had been inactivated with the same kanamycin resistance cassette used in the ybeA strain, and, in theory, xylA cells should grow at the same rate as the wild-type strain in rich medium unless expression of the resistance cassette has a biological cost.…”

“…At the end of every 6-h cycle, cells were diluted to an A 450 of 0.01 in fresh medium. The relative numbers of ybeA and xylA cells were followed throughout the growth cycles by plating on MacConkey agar supplemented with 1% (w/v) D-xylose (Toh et al 2008), and also on MacConkey/xylose agar with kanamycin at 50 mg/L, and screening for red and white colonies. In parallel experiments, the relative numbers of wild-type and ybeA cells were estimated by plating on LB agar (where all cells grow) and LB agar with kanamycin at 50 mg/L (only the ybeA cells grow).…”

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

“…m 2 A2503 is located at the entrance of the nascent peptide exit tunnel and has been involved in the ribosomal mechanism that promotes translation arrest in response to specific peptide sequences (Vazquez-Laslop et al 2010;Ramu et al 2011). Lack of methylation of A2503 has been reported to produce a subtle yet significant effect on cell fitness and to increase linezolid resistance (Toh et al 2008;Gao et al 2010;Lamarre et al 2011). RlmN and its evolutionarily related resistance enzyme Cfr belong to the radical S-adenosyl-L-methionine (SAM) enzyme superfamily, and both use protein-free rRNA as a substrate (Atta et al 2010;Kaminska et al 2010;Yan et al 2010;Grove et al 2011;Yan and Fujimori 2011).…”

Identification of pseudouridine methyltransferase in Escherichia coli

“…Although a ribosome lacking post-transcriptional modifications is able to synthesize peptides in vitro (Krzyzosiak et al 1987;Cunningham et al 1991;Green and Noller 1999;Khaitovich et al 1999), the conservation and clustering of modified nucleosides in functionally important regions of the ribosome suggest that they might be important for efficient translation, rRNA folding, ribosome assembly, or stability of ribosomes in vivo (Noller and Woese 1981;Brimacombe et al 1993;Ofengand and Fournier 1998;Decatur and Fournier 2002;Xu et al 2008). Indeed, several rRNA modifications have been shown to be important for 30S and 50S assembly and ribosome functioning (Igarashi et al 1981;Green and Noller 1996;Gustafsson and Persson 1998;Caldas et al 2000), and a number of additional modifications provide advantages under particular conditions, such as conferring resistance against ribosometargeting antibiotics (Cundliffe 1989;Weisblum 1995;Mann et al 2001;Toh et al 2008). However, the possible functional roles of the vast majority of modified nucleosides in rRNA remain unknown.…”

“…All known rRNA methyltransferases belong to class I or class IV. The genes corresponding to 16 out of the 24 rRNA methyltransferases predicted in E. coli have been identified (Andersen and Douthwaite 2006;Sergiev et al 2007Sergiev et al , 2008Toh et al 2008), and the majority of them belong to class I, characterized by the presence of a common, conserved Rossmann fold SAM binding domain (Schubert et al 2003; for review, see Ofengand and Del Campo 2004). Much less conservation is noticed at the sequence level, where only a few conserved motifs are present, most of them being a part of the SAM binding region (Fauman et al 1999).…”