Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S 150 supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ.The ribosome is a complex ribonucleoprotein particle that is responsible for translation of messenger RNAs into proteins. In Escherichia coli, it is composed of 23, 16, and 5 S ribosomal RNAs and of about 52 proteins. Twenty-one of them assemble with the 16 S rRNA to form the 30 S ribosomal subunit, while the 31 others assemble with the 23 and 5 S rRNA to form the 50 S ribosomal subunit (1). Ribosomal proteins and rRNAs cooperate both in the assembly and in the activity of the ribosome (1). The functional domains of the ribosome include a GTPase center, a peptidyl transferase center, and A-, P-, and E-tRNA binding sites; they involve specific regions of the rRNAs and one or several ribosomal proteins (1). The 16 and 23 S ribosomal RNAs and several ribosomal proteins are methylated at specific sites. The mature E. coli 16 and 23 S rRNAs have 10 and 14 methylated nucleotides, respectively (2, 3). The methyl groups are clustered at the functional domains, e.g. the A-and P-tRNA binding sites for 16 S rRNA, and the peptidyltransferase center for 23 S rRNA (4). Most of the modified nucleotides are conserved (5); however, their functions are poorly understood. It has been suggested that methylation could modulate rRNA maturation, affect stability of rRNA structures, or alter translation rates. 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. The 23 S rRNA displays 23 nucleotide modifications, of which 14 correspond to methylation. Recently, the rrmA gene encoding a 23 S rRNA methyltransferase that forms m 1 G 745 was identified (10). In the present study, we show that the heat shock protein FtsJ methylates the 50 S ribo...

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SummarySsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.