Translational Defects of Escherichia coli Mutants Deficient in the Um2552 23S Ribosomal RNA Methyltransferase RrmJ/FTSJ

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

“…3A). This observation might be explained by low translational fidelity caused by the U2252C mutation that prevents 2′-O-methylation (19), in addition to the retarded 50S assembly by this mutation. We also made a double-mutant strain, ΔrlmE/U2552C, by deleting rlmE in the U2552C strain; the resultant strain was also cold-sensitive ( Fig.…”

“…1B), which is an essential base that anchors the CCA terminus of A-site tRNA in the 50S subunit (18). The absence of a 2′-O-methyl group at Um2552 in the rlmE deletion strain of E. coli (ΔrlmE) decreases the rate of programmed frameshifting and read-through of stop codons (19), indicating that Um2552 is required for optimization of translational accuracy and thus allows natural recoding events.…”

A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain

“…We have previously suggested as one of a number of possibilities that the guide RNAs used in eukaryotes to identify the U residues destined for conversion to ⌿ might be RNA chaperones for the correct folding of ribosomal RNA (Ofengand & Fournier, 1998)+ In this view, ⌿ formation would be merely a signal that folding had occurred and it was time for the chaperone to dissociate, rather than having an intrinsic function+ If this view were extended to prokaryotes, substituting the synthases for guide RNAs, the ⌿ synthases might then be protein chaperones of RNA folding, helping in some as yet undefined way to achieve the correct structure+ However, it does not seem so likely that ⌿ formation is a necessary completion signal, as in its absence, when mutant RluD was used, complete growth rate restoration was observed (Table 2)+ More likely, RluD, and possibly the other synthases as well, have two distinct functions, one of which is related to the observed growth defects, and the other to ⌿ formation, the latter occurring for still unknown reasons+ There is some precedent for these ideas+ First, the methyltransferase that makes m 5 U54 in tRNA is indispensable in E. coli, yet its methylation activity is not required (Persson et al+, 1992)+ Second, Dim1p, the yeast enzyme which makes m 6 2 Am 6 2 A at the 39 end of the small subunit rRNA and is essential for the yeast cell (Lafontaine et al+, 1994), can dispense with its methylase activity (Lafontaine et al+, 1998)+ Third, two LSU rRNA 29-O-methyltransferases are known whose absence perturbs ribosome assembly and results in severe or lethal growth defects+ One is PET56, a yeast 29-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria (Sirum-Connolly & Mason, 1993)+ The other is FtsJ, which makes Um2552 in E. coli (Bügl et al+, 2000;Caldas et al+, 2000aCaldas et al+, , 2000b)+ In the latter example, it is not known whether the 29-O-methylation is required for proper ribosome assembly, but in the former case, recent work has shown that rescue of ribosome assembly does not require methylation (T+ Mason, pers+ comm+)+ Thus, like RluD and TruB, the protein is needed but not the product of the reaction it catalyzes+ Inhibition of 50S subunit assembly in both Dust and Tiny RluD-minus strains has also recently been observed (L+ Peil, N+ Gutgsell, J+ Ofengand, & J+ Remme, unpubl+ results)+ Considering all these results, a pattern begins to emerge in which rRNA modifying enzymes, at least the most common ones, ⌿ synthases and 29-O-methyltransferases, may have an assembly function in their own right, independent of their catalytic role in modified base formation+ In this regard, it is interesting to note the amino acid sequence homology between the N-terminus of RluD (residues 19-49) of this 326 amino acid protein with residues 10-40 of Hsp15, a protein highly induced by heat shock (Korber et al+, 1999)+ Hsp15 is a 133 amino acid protein that consists almost entirely of a new type of RNA-binding fold and which binds to 50S subunits carrying nascent protein chains with nanomolar affinity + In this context, the occurrence of pseudorevertants should be noted+ Such second-site mutants are found readily in the Dust strain, and pains must be taken to avoid them during subculturing+ They occur much more rarely in the Tiny strain, possibly because the grow...…”

The CRM domain: An RNA binding module derived from an ancient ribosome-associated protein

“…However, interpretation of these results is complicated by the fact that the ''-35'' region for the more upstream of two promoters driving expression of the adjacent, divergently transcribed ftsJ/rrmJ gene (Herman et al 1995) maps within the YhbY ORF and was deleted in this strain. RrmJ mutants show ribosome defects that resemble those in the DyhbY strain (Bugl et al 2000;Caldas et al 2000b), suggesting that reduced rrmJ expression might contribute to the DyhbY phenotype. However, RrmJ protein accumulated to near normal levels in the DyhbY strain, and introduction of an RrmJ expression plasmid into DyhbY cells did not fully restore their growth and ribosome assembly defects (data not shown).…”