S-adenosyl-L-methionine + uridine2552 in 23S rRNA = S-adenosyl-L-homocysteine + 2'-O-methyluridine2552 in 23S rRNA
mechanism, based on modeling studies and the structure of the 50S ribosome, a two-step model is proposed where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2'-O position of the ribose
identification of the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. 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
the 2'-O-ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo. A reaction mechanism for the methyltransfer activity of RrmJ is proposed
the isolated unmodified A loop serves as the minimal methylation substrate of wild-type RrmJ in vitro. 50S ribosomal subunits prepared from the rrmJ deletion strain appear to serve as substrates for RrmJ in vitro, while naked 23S rRNA or 40S ribosomal particles that are prepared from the rrmJ deletion strain are not methylated by purified RrmJ. This finding suggests that either the correct folding of the 23S rRNA or additional protein-protein interactions are necessary for the substrate recognition. A positively charged, highly conserved ridge in RrmJ appears to play a significant role in 23S rRNA binding and methylation. A structural model is provided of how the A loop of the 23S rRNA binds to RrmJ. Based on modeling studies and the structure of the 50S ribosome, a two-step model is proposed where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2'-O position of the ribose
absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit
lack of U2552 methylation, obtained in rrmJ-deficient mutants, results in a decrease in programmed +1 and -1 translational frameshifing and a decrease in readthrough of UAA and UGA stop codons. The increased translational accuracy of rrmJ-deficient strains suggests that the interaction between aminoacyl-tRNA and U2552 is important for selection of the correct tRNA at the ribosomal A site, and supports the idea that translational accuracy in vivo is optimal rather than maximal, thus pointing to the participation of recoding events in the normal cell physiology
rrmJ-deficient strain exhibit growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S30 rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity
rrmJ gene deletion causes a severe growth defect and accumulation of aberrant 50S ribosomal subunits in DELTArrmJ. Overexpression of GTPase Der suppresses growth impairment, effects of several Der mutants, overview. In a rrmJ deletion strain, two GTPase domains of Der regulate its association with 50S subunit via the KH-like domain. Phenotypes, overview
the RrmJ-catalyzed methylation of Um2552 in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures
in an Escherichia coli strain lacking SAH nucleosidase Mtn, in which cellular SAM is down-regulated, hypomodification of several methylation sites is observed, including 2'-O-methylation at position 2552 (Um2552) of 23S rRNA. There is a severe growth defect of the strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. The growth defect is partially restored by overexpression of SAM-dependent methyltransferase RlmE
in an Escherichia coli strain lacking SAH nucleosidase Mtn, in which cellular SAM is down-regulated, hypomodification of several methylation sites is observed, including 2'-O-methylation at position 2552 (Um2552) of 23S rRNA. There is a severe growth defect of the strain with significant accumulation of 45S ribosomal precursor harboring 23S rRNA with hypomodified Um2552. The growth defect is partially restored by overexpression of SAM-dependent methyltransferase RlmE
identification of a conserved tetrad K-D-K-H in the family of small nucleolar RNA-guided ribose 2'-O-methyltransferases related to fibrillarin. The corresponding functional groups of putative catalytic tetrads of Escherichia coli RrmJ and Methanococcus jannaschii Mj0697 may be superimposed in space. The invariant residues K164 in RrmJ and K179 in Mj0697 are observed in two distinct locations in the primary sequence, suggesting an interesting case of migration of the conserved side chain within the framework of the active site
identification of a conserved tetrad K-D-K-H in the family of small nucleolar RNA-guided ribose 2'-O-methyltransferases related to fibrillarin. The corresponding functional groups of putative catalytic tetrads of Escherichia coli RrmJ and Methanococcus jannaschii Mj0697 may be superimposed in space. The invariant residues K164 in RrmJ and K179 in Mj0697 are observed in two distinct locations in the primary sequence, suggesting an interesting case of migration of the conserved side chain within the framework of the active site
identification of a conserved tetrad K-D-K-H in the family of small nucleolar RNA-guided ribose 2'-O-methyltransferases related to fibrillarin. The corresponding functional groups of putative catalytic tetrads of Escherichia coli RrmJ and Methanococcus jannaschii Mj0697 may be superimposed in space. The invariant residues K164 in RrmJ and K179 in Mj0697 are observed in two distinct locations in the primary sequence, suggesting an interesting case of migration of the conserved side chain within the framework of the active site
the mutant D124A is unable to rescue the growth defect of the rrmJ deletion strain, indicating that this mutation causes the inactivation of RrmJ in vivo
the mutant D83A is unable to rescue the growth defect of the rrmJ deletion strain, indicating that this mutation causes the inactivation of RrmJ in vivo
the RrmJ deletion strains expressing the E199A variant protein shows only slight growth defects, indicating that the residue is not as important in the catalytic mechanism
the mutant D83A is unable to rescue the growth defect of the rrmJ deletion strain, indicating that this mutation causes the inactivation of RrmJ in vivo
the mutant D83A is unable to rescue the growth defect of the rrmJ deletion strain, indicating that this mutation causes the inactivation of RrmJ in vivo
R32A/R34A mutant strain accumulates larger amounts of 30S and 50S ribosomal subunits than wild-type strains under nonstringent salt conditions, and has a significant amount of 40S ribosomal particles under stringent salt conditions
the RrmJ deletion strains expressing the Y201A variant protein shows only slight growth defects, indicating that the residue is not as important in the catalytic mechanism
D136N mutant strain accumulates larger amounts of 30S and 50S ribosomal subunits than wild-type strains under nonstringent salt conditions, and has a significant amount of 40S ribosomal particles under stringent salt conditions
lack of U2552 methylation, obtained in rrmJ-deficient mutants, results in a decrease in programmed +1 and -1 translational frameshifing and a decrease in readthrough of UAA and UGA stop codons. The increased translational accuracy of rrmJ-deficient strains suggests that the interaction between aminoacyl-tRNA and U2552 is important for selection of the correct tRNA at the ribosomal A site, and supports the idea that translational accuracy in vivo is optimal rather than maximal, thus pointing to the participation of recoding events in the normal cell physiology
rrmJ-deficient strain exhibit growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S30 rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity
rrmJ-deficient strain exhibit growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S30 rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity