The enzyme efficiently methylates guanine966 of the assembled 30S subunits in vitro. Protein-free 16S rRNA is not a substrate for RsmD . The enzyme specifically methylates guanine966 at N2 in 16S rRNA.
The enzyme efficiently methylates guanine966 of the assembled 30S subunits in vitro. Protein-free 16S rRNA is not a substrate for RsmD [1]. The enzyme specifically methylates guanine966 at N2 in 16S rRNA.
the enzyme uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations shows that S7 plus S19 are sufficient to block methylation by the m5C967 methyltransferase, while simultaneously inducing methylation by the m2G966 methyltransferase. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA is isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G966 methyltransferase but not by the m5C967 methyltransferase. Since binding of S19 requires prior binding of S7, which has no effect on methylation when bound alone, the switch in methylase specificity is attributed solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 results in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G966 methyltransferase, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C967 methyltransferase. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides
RsmD methyltransferase utilizes assembled small subunits or its late assembly intermediates as a substrate. It is likely that the latter class of proteins uses the decoding cleft of the small subunit as the binding site
RsmD protein efficiently methylates guanine966 of the assembled 30S subunits (purified from yhhF knock-out strain in vitro) in vitro in the presence of AdoMet. Protein-free 16 S rRNA was not a substrate for RsmD. The methylation is specific for guanine966 of 16S rRNA
the N-terminal domain of Rv2966c is important for binding not only RNA but also DNA substrates, binding of protein to DNA is much stronger than it is to RNA
the N-terminal domain of Rv2966c is important for binding not only RNA but also DNA substrates, binding of protein to DNA is much stronger than it is to RNA
the enzyme uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations shows that S7 plus S19 are sufficient to block methylation by the m5C967 methyltransferase, while simultaneously inducing methylation by the m2G966 methyltransferase. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA is isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G966 methyltransferase but not by the m5C967 methyltransferase. Since binding of S19 requires prior binding of S7, which has no effect on methylation when bound alone, the switch in methylase specificity is attributed solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 results in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G966 methyltransferase, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C967 methyltransferase. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides
Structural and functional characterization of Rv2966c reveals an RsmD-like methyltransferase from M. tuberculosis and the role of its N-terminal domain in target recognition.
disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at guanine966. Doubling of strain JW3430 carrying the inactive rsmD gene is essentially the same as that of the parental Escherichia coli strain BW25141
truncated constructs of Rv2966c lacking the N-terminal 18- or 23-amino acid residues and the mutant protein 3RM do not show any significant binding to either DNA or RNA nor any m2G966 methyltransferase activity
RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation
based on a comprehensive bioinformatic analysis of m2G methyltransferases it is inferred that the prokaryotic RsmC and RsmD methyltransferases are pseudodimers. The C-terminal catalytic domain is closely related to the structurally characterized Mj0882 protein, while the N-terminal domain lacks the cofactor-binding and catalytic side-chains
protein Rv2966c shows a two-domain structure with a short hairpin domain at the N-terminus and a C-terminal domain with the S-adenosylmethionine-methyltransferase-fold. The N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. In contrast to the variable substrate binding domain, the C-terminal domain is a highly conserved structure consisting of the classical S-adenosyl-L-methionine-methyltransferase-fold found in several S-adenosyl-L-methionine-dependent methyltransferases. It consists of a central eight-stranded beta-sheet flanked by alpha-helices on both sides, the first five strands of the beta-sheet are parallel to each other and encompass the S-adenosyl-L-methionine binding site toward their C terminus end, whereas the remaining strands of the beta-sheet are antiparallel. The N-terminus of Rv2966c is the target recognition region that helps in binding to nucleic acids
protein Rv2966c shows a two-domain structure with a short hairpin domain at the N-terminus and a C-terminal domain with the S-adenosylmethionine-methyltransferase-fold. The N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. In contrast to the variable substrate binding domain, the C-terminal domain is a highly conserved structure consisting of the classical S-adenosyl-L-methionine-methyltransferase-fold found in several S-adenosyl-L-methionine-dependent methyltransferases. It consists of a central eight-stranded beta-sheet flanked by alpha-helices on both sides, the first five strands of the beta-sheet are parallel to each other and encompass the S-adenosyl-L-methionine binding site toward their C terminus end, whereas the remaining strands of the beta-sheet are antiparallel. The N-terminus of Rv2966c is the target recognition region that helps in binding to nucleic acids
purified recombinant Rv2966c, hanging drop vapour diffusion method, mixing of 0.004 ml of 17 mg/ml Rv2966c in 10 mM potassium phosphate buffer, pH 7.4, 5 mM EDTA, 10% v/v glycerol, 0.1% v/v 2-mercaptoethanol, 50 mM NaCl, 0.1 mM S-adenosyl-L-methionine, with 0.002 ml of reservoir solution containing 100 mM Tris-HCl, pH 7.4, 3.0 M potassium acetate at 24 °C, 2-3 days, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement
site-directed mutagenesis, the mutant shows increased Km toward the 30S subunit by two orders of magnitude with only a marginal effect on kcat compared to the wild-type enzyme. The mutation decreases the enthalpic contribution to SAM binding, paralleled by an increase in the entropic contribution
site-directed mutagenesis, the mutant shows increased Km toward the 30S subunit by two orders of magnitude with only a marginal effect on kcat compared to the wild-type enzyme
recombinant Rv2966c with His10-Smt3 fusion at the N-terminus from Escherichia coli strain BL21 (DE3) by nickel affinity and anion exchange chromatography
gene rsmD, complementation of rsmD-deleted Escherichia coli KL16DELTArsmD cells, expression of Rv2966c with His10-Smt3 fusion at the N-terminus from plasmid pMTase1 in Escherichia coli strain BL21 (DE3)
Rychlewski. L.: RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited--bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure
Weitzmann, C.; Tumminia, S.J.; Boublik, M.; Ofengand, J.
A paradigm for local conformational control of function in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of 57 is required for methylation of m2G966 and blocks methylation of m C967 by their respective methyltransferases
Kumar, A.; Saigal, K.; Malhotra, K.; Sinha, K.M.; Taneja, B.
Structural and functional characterization of Rv2966c protein reveals an RsmD-like methyltransferase from Mycobacterium tuberculosis and the role of its N-terminal domain in target recognition
Arora, S.; Bhamidimarri, S.P.; Weber, M.H.; Varshney, U.
Role of the ribosomal P-site elements of m2G966, m5C967, and the S9 C-terminal tail in maintenance of the reading frame during translational elongation in Escherichia coli
Arora, S.; Bhamidimarri, S.P.; Bhattacharyya, M.; Govindan, A.; Weber, M.H.; Vishveshwara, S.; Varshney, U.
Distinctive contributions of the ribosomal P-site elements m2G966, m5C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli