N2 -Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m 2 G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m 2 G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m 2 G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å . The structure closely resembles RsmC rRNA methyltransferase, specific for m 2 G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m 2 G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.Ribosomal RNAs form the main functional core of the ribosome, a universal molecular machine for protein synthesis. It is believed that primordial ribosome was initially composed entirely of RNA (1) and proteins were added later in evolution.A set of four ribonucleotides used in rRNA is rather limited for the full range and fine tuning of ribosomal functions especially when compared with 20 amino acids the proteins are composed of. The diversity in RNA could be increased through the usage of a set of modified nucleotides (2). A number of such nucleotides have been found in functional ribosomal RNA molecules (as well as other RNA). It was also shown that nucleotide modification is an important component of the ribosome maturation process (3). The nucleotide modifications can involve bases and ribose. The most common in bacteria is base methylation, but other substitutions have also been reported (4). The specific functional role of nucleotide modifications is not always obvious, but their clustering in the ligand-binding and catalytic centers (5) suggests their involvement in the vital ribosome functions. Even more striking is the fact that many of the nucleotide methylations are performed by site-specific RNA methyltransferases, at a rather significant metabolic cost to the cell.The surrounding of the P-site of the ribosome is particularly rich in modified bases. Several RNA small patches consisting entirely of modified bases have been found. For example, helix 31 of 16 S rRNA contains two modified bases in a row: m 2 G966 and m 5 C967 (Fig. 1A). m 2 G966 is in direct contact with P-sitebound tRNA as revealed by a footprinting assay (6) and later structural studies (7,8). It is located in the head of the small ribosomal subunit, above the P-site-bound tRNA and forms a direct contact with the tip of the anticodon l...

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.

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Escherichia coli ribosomal RNA contains five guanosine residues methylated at N2. The ygjO gene was previously predicted to methylate 16 S rRNA residue G966 due to its high sequence homology with the protein RsmC, responsible for G1207 methylation. We have identified the target of YgjO as being m2G1835 of the 23 S rRNA and not m2G966 of the 16 S rRNA as expected. Knock-out of the ygjO gene leads to loss of modification at G1835, as revealed by reverse transcription. Moreover, the modification could be restored by in vivo complementation of the ygjO knock-out strain with a plasmid expressing ygjO. Recombinant YgjO protein is able to methylate in vitro protein-free 23 S rRNA, but not assembled 50 S subunits purified from the ygjO knock-out strain. The nucleotide m2G1835 is located in a functionally extremely important region of the ribosome, being located within intersubunit bridges of group B2. Growth competition assays reveal that the lack of the G1835 methylation causes growth retardation, especially at temperatures higher than optimal and in poor media. Based on these results we suggest that YgjO be renamed to RlmG in accordance with the accepted nomenclature for rRNA methyltransferases.