small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation
the enzyme is highly specific for U1498 in 16S rRNA. The purified enzyme is able to incorporate methyl groups from S-adenosyl-L-methionine into 30S particles purified from the mutant strain but not into 30S particles purified from wild type. Approximately one methyl group could be incorporated per mutant 30S subunit at high levels of enzyme
RsmE requires a highly structured ribonucleoprotein particle (a fully assembled 30S ribosome subunit) as a substrate for methylation, and this methylation occurs late during 30S ribosome assembly
RsmE requires a highly structured ribonucleoprotein particle (a fully assembled 30S ribosome subunit) as a substrate for methylation, and this methylation occurs late during 30S ribosome assembly
most active in the presence of 1015 mM Mg2+ at pH 79. At 30 mM Mg2+ optimal activity is reduced by 60%. In the presence of spermidine, Mg2+ is not required for activity. Activity could be restored up to 60% of that with Mg2+ by addition of spermidine at 30 mM
in the presence of spermidine, Mg2+ is not required for activity. Activity could be restored up to 60% of that with Mg2+ by addition of spermidine at 30 mM
RsmE is the founding member of a RNA methyltransferase family responsible for N3-methylation of U1498 in 16S ribosomal RNA. It is well conserved across bacteria and plants and may play an 30 important role in ribosomal intersubunit communication
a yggJ deletion strain lacks the methyl group at U1498 as well as the specific methyltransferase activity. The deletion strain is unaffected in exponential growth in rich or minimal media at multiple temperatures, but it is defective when grown in competition with isogenic wild-type cells
RsmE forms a flexible dimeric conformation that is essential for substrate binding. RsmE-S-adenosyl-L-methionine-uridylic acid complex modeling and substrate binding structure, overview. The MTase domain of one subunit in dimeric RsmE is responsible for binding of one S-adenosyl-L-methionine molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule, the ribosomal RNA fragment and ribosomal protein complex. The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. Molecular replacement of crystal structure, PDB ID 1VHY
the crystal structure in monomer shows that RsmE consists of two distinct but structurally related domains: the pseudouridine synthases and archaeosine-specific transglycosylasess-like RNA recognition and binding domain, PUA, and the conserved MTase domain with a deep trefoil knot
the methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. Molecular replacement of crystal structure, PDB ID 1VHY. Structural comparisons of N-terminal and C-terminal domains of RsmE with related proteins, overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method at room temperature, 0.15 M potassium thiocyanate and 24% w/v PEG monomethyl ether 2000, 3-4 days, crystal soaking in S-adenosyl-L-methionine solution or cocrystallization of enzyme and cofactor are not successful, X-ray diffraction structure determination and analysis at 2.25 A resolution, molecular replacement
the deletion of critical residues at the C-terminus of Rv2372c leads to an inability of the protein to form stable dimers and to abolition of the methyltransferase activity
the deletion of critical residues at the C-terminus of Rv2372c leads to an inability of the protein to form stable dimers and to abolition of the methyltransferase activity
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2.1.1.193 16S rRNA (uracil1498-N3)-methyltransferase
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