Mutations in 16S rRNA in<i>Escherichia coli</i>at methyl-modified sites: G966, C967, and G1207

Jemiolo, David K.; Taurence, Jacqueline S.; Giese, Sharon Y.

doi:10.1093/nar/19.15.4259

Cited by 31 publications

(24 citation statements)

References 23 publications

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“…RsmD ortholog distribution agrees with conservation of its target nucleotide. In agreement with the lack of conservation mutations of m 2 G966 in E. coli, this does not lead to significant growth retardation (10). Mutant 30 S subunits are normally incorporated into the 70 S ribosome pool and show no functional defects.…”

Section: Discussionsupporting

confidence: 75%

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Methyltransferase That Modifies Guanine 966 of the 16 S rRNA

Lesnyak

Osipiuk

Skarina

et al. 2007

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 75%

“…1B). Interestingly, despite the lack of conservation of the nucleotide 966 among the kingdoms of life (9), and phenotypically silent character of its mutants (10), the modified nature of this base is conserved (11,12). A protein with RNA methyltransferase activity, specific for m 2 G966, was partially purified from Escherichia coli extracts (13).…”

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confidence: 99%

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA

Lesnyak

Osipiuk

Skarina

et al. 2007

Journal of Biological Chemistry

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“…Mutations in this region may affect protein synthesis (10), either by a change in binding of tRNA to the P site itself or by blocking the conformational change needed for the tRNA binding to the A site. In H. pylori strain 181 and the Tet r transformants of strain FIG.…”

Section: Discussionmentioning

confidence: 99%

16S rRNA Mutation-Mediated Tetracycline Resistance in Helicobacter pylori

Gerrits

Zoete

Arents³

et al. 2002

Antimicrob Agents Chemother

116

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Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet r ) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Helicobacter pylori is a spiral-shaped, gram-negative bacterium that causes chronic infections in the gastric mucosa (6). This infection will persist for life, unless treated with antibiotics. Cure of H. pylori infection results in ulcer healing and may reduce the risk of gastric cancer and gastric lymphoma (22,28). The highest cure rates have been obtained with antimicrobial treatments that include two or more antimicrobial drugs, a bismuth component, and/or a proton pump inhibitor (14, 25). For the treatment of H. pylori infections, tetracycline-based triple or quadruple therapies are often used as a second-line treatment (7,9,17). Until the end of the last century only a few reports were published on spontaneous tetracycline resistance (18; P. D. Midolo, M. G. Korman, J. D. Turnidge, and J. R. Lambert, Letter, Lancet 347:1194-1195, 1996), and it was generally accepted that tetracycline resistance (MIC Ն 4 g/ml) in H. pylori is very rare (5, 12). However, in the last 2 years an increase in the incidence of tetracycline resistance in H. pylori has been reported (2,11,13,20,30).Tetracycline inhibits the protein synthesis by binding to the 30S ribosomal subunit (3,19). In most bacteria resistance to tetracycline is due to an energy-dependent efflux of tetracycline-cation complexes across the cell membrane by membrane-associated efflux proteins. Export of tetracycline complexes out of the cell reduces the intracellular drug concentration and protects the ribosomes from tetracycline (4). Overexpression of the efflux genes confers tetracycline resistance, while the sensitivity to tetracycline increases by deletions in these genes. The second common mechanism of resistance is mediated through ribosomal protection proteins. These cytoplasmic proteins confer tetracycline resistance either by a reduction of the affinity of ribosomes for tetracycline or by releasing the bound antibiotic from the ribosome. The ribosomal protection proteins, such as TetM, TetO, and TetS, show homology with the elongation factors EF-G and EF-Tu (Table 1) (4). Beside these two most common tetracycline resistance mechanisms, two other mechanisms have been described. One is based on enzymatic inactivation of tetracycline by the product of TetX in the presence of oxygen and NADPH, and the other originates from mutations in the 16S rRNA ge...

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“…The main plasmids used in this study were as follows. pc1857 was derived from pACYC177 and contains a temperature-sensitive bacteriophage A repressor gene (12). pDJ100 was derived from pBR322 and contains the wild-type rRNA operon, rrnB, under the control of the A PL promoter.…”

Section: Methodsmentioning

confidence: 99%

UGA suppression by a mutant RNA of the large ribosomal subunit.

Jemiolo

Pagel²,

Murgola³

1995

Proc. Natl. Acad. Sci. U.S.A.

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A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 16S rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA(UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L1I, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 16S rRNA (small subunit) mutant ofEscherichia coli that suppressed UGA mutations and implicated in particular the universally conserved nucleotide C1054, located in the conserved helix 34 (1). A key role for 23S rRNA (large ribosomal subunit) in peptide bond formation, translocation, and translational accuracy has been clearly established (refs. 2-5 and references therein). The small ribosomal subunit, on the other hand, has generally been thought to be the subunit involved in codon-specific translational events in both elongation and termination. Indeed, until recently, the majority of rRNA suppressors of nonsense (termination codon) mutations obtained were associated with 16S rRNA (6). In this paper, however, we describe a 23S rRNA nonsense suppressor that works at UGA mutant codons in trpA but not at UAA or UAG mutations at the same codon positions. A preliminary report of these results was presented at the Cold Spring Harbor Laboratory meeting on the Mo-

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Mutations in 16S rRNA inEscherichia coliat methyl-modified sites: G966, C967, and G1207

Cited by 31 publications

References 23 publications

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA

16S rRNA Mutation-Mediated Tetracycline Resistance in Helicobacter pylori

UGA suppression by a mutant RNA of the large ribosomal subunit.

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