Mechanism of protein synthesis inhibition by fusidic acid and related antibiotics

An RNA-protein complex consisting of 5S RNA and two ribosomal proteins, B-L5 and B-L22, was isolated from Bacillus stearothermophilus ribosomes and found to be active in GTP hydrolysis. This activity was not influenced by elongation factor G. Further analysis of this complex showed that it was also able to hydrolyze ATP. Inhibition studies revealed that ATP was a noncompetitive inhibitor of GTP and that GTP was also a noncompetitive inhibitor of ATP, indicating that two different enzymatic sites were involved. Differences in pH optimum and optimal temperature also point to a twosite enzyme complex. Both enzymatic hydrolyses were inhibited by thiostrepton and fusidic acid, which are known inhibitors of protein synthesis.In order to accomplish protein synthesis, ribosomes perform an intricate pattern of enzymatic activities (for recent reviews on the structure and function of bacterial ribosomes see refs.

Section: Resultsmentioning

confidence: 99%

ATPase and GTPase Activities Associated with a Specific 5S RNA-Protein Complex

Horne

Erdmann

1973

“…Inhibition by fusidic acid is a common property shared by the ribosome-dependent GTPase reaction in either eukaryotic or bacterial systems (17)(18)(19)(20)(21). We tested our GTPase factor-dependent hydrolysis and found that 2 Diphtheria toxin-catalyzed transfer of ADP-ribose from NAD to EF-2 provides a highly specific way of measuring EF-2 in the absence of any other protein synthetic component (8)(9)(10)(11) (1) CL not carry out identical functions.…”

Section: Methodsmentioning

confidence: 99%

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

Skogerson¹,

Wakatama²

1976

Three proteins required for poly(U)directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-. catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. Ribosomal functions in vitro require the participation of specific soluble proteins that are readily removed by standard isolation procedures (1). Two such proteins, the elongation factors (EF), are required for the poly(U)-directed formation of polyphenylalanine. One factor participates in codon recognition by binding the correct aminoacyl-tRNA to ribosomes while the other factor is required for translocation. Binding factor from eukaryotic cells, EF-1, is functionally very similar to that from bacteria, EF-Tu. The bacterial EF-G appears analogous to EF-2 in that both are required for translocation (2, 3).In the course of purifying elongation factors from the yeast, Saccharomyces ceretsiae, we have identified and partially purified a third protein with elongation factor activity. Our studies show that the third factor is required, in addition to EF-1 and EF-2, for poly(U)-directed polyphenylalanine synthesis. In examining possible functions for this new factor we found that it promoted the ribosome-dependent hydrolysis of GTP. On the other hand EF-2 had only a small effect on ribosome-dependent GTP hydrolysis, in either the presence or absence of the third factor. T-500, 339 ml of 30% (weight/weight) polyethylene glycol 6000, and sufficient NH4Cl to give a final concentration of 0.1 M. The suspension was stirred for 30 min and the phases were separated by centrifugation at 5000 X g for 10 mmin. The upper polyethylene glycol phase contained about 30-409k of the protein, but negligible elongation factor activity and was discarded. To each 100 ml of the lower Dextran phase was added 247 ml of buffer A, 82 ml of the 30% polyethylene glycol solution, and sufficient NH4Cl to give a final concentration of 2.0 M. The polyethylene glycol phase was recovered as before (2 M polyethylene glycol) and was treated with ammonium sulfate as previously described (4) to remove the bulk of the polyethylene glycol. To each 1000 ml of the 2 M polyethylene glycol fraction 144 g of solid ammonium sulfate was added (25% saturation). When the salt had dissolved, the phases were centrifuged at 5000 X g for 10 min and the lower phase was removed by aspiration. Proteins were precipitated from this solution by adding 267 g of ammonium sulfate per 1000 ml (65% saturation) of solution. The precipitate was collected by centrifugation, dissolved in buffer B (0.1 M potassium phosphate, pH 6.8, 10% glycerol, and 10 mM 2-mercaptoethanol), and desalted on a Sephadex G-25 column equilibrated with the same buffer.Hydroxylapatite Chromatography. Hydroxylapatite (Bio-Rad, HT) was packed in a...

“…Indeed, the continued rise of resistance to antibiotics in this species has rendered FA one of the few remaining oral agents available for treatment of infections caused by methicillin-resistant S. aureus (MRSA) (4). FA inhibits bacterial protein synthesis by binding to the ribosomal translocase, elongation factor G (EF-G), when the latter is associated with the ribosome (5,6). FA does not interfere with the primary catalytic function of EF-G, which involves the translocation of mRNA and associated tRNAs on the ribosome in a reaction coupled to the hydrolysis of GTP (7).…”

mentioning

confidence: 99%

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Cox

Thompson

Jenkins

et al. 2012

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusBtype proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FAmediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome·EF-G·GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.antibacterial | protein synthesis | translocation | structure