Abstract
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.
Highlights
Despite their specificity and potency, the widespread use of these antibiotics by humans has spurred the spread of resistant strains of pathogenic bacteria [1]
To explore the biochemical nature of Fusidic acid-resistant (FusR) elongation factor G (EF-G) proteins, in this study, we examined the functions of highly conserved residues in the interdomain pocket of elongation factor (EF)-G that are mutated in bacteria most resistant to fusidic acid
FusR Mutants in E. coli EF-G—We selected seven residues of EF-G that are mutated in bacteria of clinical and laboratory origins that display the highest levels of resistance to fusidic acid (4 – 8)
Summary
Materials—Ribosomes were purified from Escherichia coli MRE600 cells, and other materials were obtained as described [17, 18]. Reactant and product (␥-32P-labeled GTP and inorganic phosphate) were separated by TLC and quantified to calculate the values indicated on the y axis. These values were normalized for hydrolyzed GTP in the stock (between 3 and 4%). 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, tRNAfMet bound in the ribosome P site, and tRNAPhe in the ribosome A site. In fluorescent mRNA assays, pyrene was attached to the mRNA 3Ј-end; EF-G1⁄7GTP and pretranslocational ribosomes were rapidly mixed together via a stopped-flow device; and ribosome translocation was detected by quenching of the fluorescence intensity as pyrene entered the ribosome
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