Methyltransferase That Modifies Guanine 966 of the 16 S rRNA

Abstract

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. 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 2.05 Å . 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.Ribosomal RNAs form the main functional core of the ribosome, a universal molecular machine for protein synthesis. It is believed that primordial ribosome was initially composed entirely of RNA (1) and proteins were added later in evolution.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. The diversity in RNA could be increased through the usage of a set of modified nucleotides (2). A number of such nucleotides have been found in functional ribosomal RNA molecules (as well as other RNA). It was also shown that nucleotide modification is an important component of the ribosome maturation process (3). The nucleotide modifications can involve bases and ribose. The most common in bacteria is base methylation, but other substitutions have also been reported (4). The specific functional role of nucleotide modifications is not always obvious, but their clustering in the ligand-binding and catalytic centers (5) suggests their involvement in the vital ribosome functions. Even more striking is the fact that many of the nucleotide methylations are performed by site-specific RNA methyltransferases, at a rather significant metabolic cost to the cell.The surrounding of the P-site of the ribosome is particularly rich in modified bases. Several RNA small patches consisting entirely of modified bases have been found. For example, helix 31 of 16 S rRNA contains two modified bases in a row: m 2 G966 and m 5 C967 (Fig. 1A). m 2 G966 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 l...

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

“…21 Interestingly, Lesnyak et al recently showed that disruption of the m 2 G966 methyltransferase gene, rsmD, with a kanamycin resistance marker has little effect on the viability or growth rate of E. coli though the knockout strains were slightly less able to compete with wild-type cells when grown in rich medium. 22 Abdi and Fredrick, however, found that any mutation at position 966 resulted in a loss of ribosomal function (see Discussion). 23 Crystal structures of Thermus thermophilus 30S subunits 24 show interactions between the 970 loop and ribosomal proteins S9, S10, and S13.…”

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

“…Here, we have identified and characterized Rv2966c as an RsmD-like methyltransferase from M. tuberculosis by genome data base search, structural, and biochemical analysis. RsmD has m 2 G966 methyltransferase activity for 16 S rRNA in E. coli (14). We have shown that rv2966c can complement rsmD-deleted E. coli cells.…”

Expanding the Nucleotide Repertoire of the Ribosome with Post-Transcriptional Modifications

“…In bacteria, nucleotide 966 is predominately a guanosine, but occupied by acp 3 U in archaea (37) and m 1 acp 3 Ψ in eukarya (38). An E. coli strain lacking the G966 modification reveals only a moderate growth disadvantage compared with the wild-type strain (39); however, recent studies have shown that specific mutations at that locus cause mistranslation (P. Cunningham, A. Saraiya, and T. Lamichhane, Wayne State University, personal communication).…”