Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor, Roxana; Murataliev, Marat B.; Wilson, Kevin S.

doi:10.1074/jbc.m707179200

Cited by 30 publications

(49 citation statements)

References 40 publications

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“…The biogenesis of the 30S ribosomal subunit involves numerous enzymes, including RNases, RNA chaperones and helicases, modifying enzymes, and small GTPases (9,12). Some of these proteins have not been identified yet, while the function of others is poorly understood.…”

Section: Discussionmentioning

confidence: 99%

The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

2010

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The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.Many heat shock proteins are chaperones and proteases that constitute the protein quality control system (4,5,13,18). Recent studies demonstrated that beyond protein quality control, the heat shock response includes proteins implemented in the translation machinery (16,17), such as FtsJ (2, 3) and Hsp15 (11).FtsJ catalyzes methylation of U2552 in 23S rRNA (3). This modification occurs during the final steps of 50S biogenesis and is important for the structural stability of the 50S subunit (2). ftsJ deletion mutants accumulate ribosomal subunits at the expense of polysomes (2). Consequently, crude ribosome extracts prepared from ftsJ deletion mutants are far less active than wild-type preparations (3). Hsp15 recognizes and binds with high affinity to the aberrant state of the 50S subunit in complex with peptidyl tRNA positioned at the A site (10), which is more frequent at high temperatures (10). It has been proposed that Hsp15 participates in releasing the bound peptide and thereby helps recycle the 50S subunit (8, 10). Thus, heat shock proteins play a significant role both in the biogenesis of ribosomes and in the translation process.YbeY is a 17-kDa heat shock protein, highly conserved among bacteria, that belongs to the UPF0054 family of metaldependent hydrolases, suggesting that it may have a potential hydrolytic function (14,21). In Aquifex aeolicus, analysis of YbeY structure homology showed similarity to eukaryotic extracellular proteinases such as collagenase and gelatinase. However, in vitro experiments could not detect collagenase, gelatinase, or other hydrolase activity in YbeY (14).Recently, we showed that ybeY deletion mutants exhibit severe translational defects manifested by a very low level of polysomes and accumulation of free ribosomes and ribosomal subunits, indicating that most ribosomes in the cell are not engaged in translation. This translational defect intensifies at elevated temperatures (42°C) and results in growth arrest (17).Here we present in vitro studies indicating that the activity of the translation machinery prepared from ybeY deletion mutants is lower than in the wild type. In addition, we show that this lower activity stems specifically from a defective 30S ribosomal subunit. MATERIALS AND METHODSPurification of ribosomes and S100 subunits. Ribosomes and S100 subunits were prepared essentially as described previously (1). Cultures of Escherichia coli MG1655 and its ⌬ybeY derivative were grown in LB broth (Difco) medium at 37°C to an optical density at 60...

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Section: Discussionmentioning

confidence: 99%

The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

2010

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“…17,18 Research by Nechifor and Wilson indicated that helix A of EF-G domain G' is directly involved in ribosome-activated GTP hydrolysis and thereby exerts indirect effects on translocation. 19 Mutating the conserved residue 224 or 228 in helix A of Escherichia coli EF-G (corresponding to amino acids 255 and 259 in human EFG1), thus disrupting electrostatic interactions with L7/L12, resulted in severe defects in GTP hydrolysis in the presence of the ribosome (without effects on basal GTPase activity) and smaller defects in ribosomal translocation. Hence, the Arg250Trp mutation in EFG1 reported in the current study is presumed to hamper ribosome-dependent GTP hydrolysis, causing impairments in mitochondrial translation and oxidative phosphorylation and consequently fatal mitochondrial encephalopathy.…”

Section: Discussionmentioning

confidence: 99%

Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

et al. 2010

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The mitochondrial translation system is responsible for the synthesis of 13 proteins required for oxidative phosphorylation (OXPHOS), the major energy-generating process of our cells. Mitochondrial translation is controlled by various nuclear encoded proteins. In 27 patients with combined OXPHOS deficiencies, in whom complex II (the only complex that is entirely encoded by the nuclear DNA) showed normal activities, and mutations in the mitochondrial genome as well as polymerase gamma were excluded, we screened all mitochondrial translation factors for mutations. Here, we report a mutation in mitochondrial elongation factor G1 (GFM1) in a patient affected by severe, rapidly progressive mitochondrial encephalopathy. This mutation is predicted to result in an Arg250Trp substitution in subdomain G' of the elongation factor G1 protein and is presumed to hamper ribosome-dependent GTP hydrolysis. Strikingly, the decrease in enzyme activities of complex I, III and IV detected in patient fibroblasts was not found in muscle tissue. The OXPHOS system defects and the impairment in mitochondrial translation in fibroblasts were rescued by overexpressing wild-type GFM1, establishing the GFM1 defect as the cause of the fatal mitochondrial disease. Furthermore, this study evinces the importance of a thorough diagnostic biochemical analysis of both muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as enzyme deficiencies can be selectively expressed.

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“…Materials-Ribosomes were purified from Escherichia coli MRE600 cells, and other materials were obtained as described (17,18). Mutations of desired EF-G codons were introduced genetically into a plasmid encoding E. coli EF-G with a C-terminal hexahistidine tag (17).…”

Section: Methodsmentioning

confidence: 99%

“…Ribosome Translocation-Translocation of the ribosome by one codon along an mRNA was monitored by assays of toeprinting and fluorescent mRNA (18). In both assays, pretranslocational ribosome complexes were formed with the E. coli ribosome containing T4 gene 32 mRNA, tRNA fMet bound in the ribosome P site, and tRNA Phe in the ribosome A site.…”

Section: Ef-g Binding and Hydrolysis Of 2ј3ј-onј-methylanthraniloylmentioning

confidence: 99%

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A Central Interdomain Protein Joint in Elongation Factor G Regulates Antibiotic Sensitivity, GTP Hydrolysis, and Ribosome Translocation

Ticu

Murataliev

Nechifor

et al. 2011

Journal of Biological Chemistry

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The antibiotic fusidic acid potently inhibits bacterial translation (and cellular growth) by lodging between domains I and III of elongation factor G (EF-G) and preventing release of EF-G from the ribosome. We examined the functions of key amino acid residues near the active site of EF-G that interact with fusidic acid and regulate hydrolysis of GTP. Alanine mutants of these residues spontaneously hydrolyzed GTP in solution, bypassing the normal activating role of the ribosome. A conserved phenylalanine in the switch II element of EF-G was important for suppressing GTP hydrolysis in solution and critical for catalyzing translocation of the ribosome along mRNA. These experimental results reveal the multipurpose roles of an interdomain joint in the heart of an essential translation factor that can both promote and inhibit bacterial translation.

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Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Cited by 30 publications

References 40 publications

The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

A Central Interdomain Protein Joint in Elongation Factor G Regulates Antibiotic Sensitivity, GTP Hydrolysis, and Ribosome Translocation

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