Abstract
N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.
Highlights
N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket
A set of four ribonucleotides used in rRNA is rather limited for the full range and fine tuning of ribosomal functions especially when compared with 20 amino acids the proteins are composed of
It is located in the head of the small ribosomal subunit, above the P-site-bound tRNA and forms a direct contact with the tip of the anticodon loop [7, 8] (Fig. 1B)
Summary
M2G966 is in direct contact with P-sitebound tRNA as revealed by a footprinting assay [6] and later structural studies [7, 8] It is located in the head of the small ribosomal subunit, above the P-site-bound tRNA and forms a direct contact with the tip of the anticodon loop [7, 8] (Fig. 1B). RNA methyltransferase, specific for C967 methylation, was shown to modify naked 16 S rRNA but not 30 S subunits [13]. RsmB methyltransferase [20], specific for the methylation of In vitro methylation assay was performed in a buffer containing adjacent residue with different base specificity (m5C967), made 50 mM HepesK, pH 7.6, 2.5 mM Mg(OAc) mM NH4Cl, 100 it clear why RsmB modify protein-free 16 S rRNA, while RsmD mM KCl, 8 mM -mercaptoethanol, 0.2 mM AdoMet. We used acts on the assembled 30 S subunit
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