The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants.
We have previously identified 20 different fusidic acid-resistant alleles of fusA, encoding mutant forms of the ribosomal translocase EF-G. One of these, P413L, is used here as the starting point in selections for internal revertants, identifying 20 different pseudo-wild-type forms of EF-G. We have also identified two alleles of fusA previously isolated as suppressors of 4.5 S RNA deficiency. All of these mutants are analysed in terms of their effects on the structural dynamics of EF-G. Most mutation conferring fusidic acid-resistance interfere with conformational changes of EF-G, but some may be located at a possible fusidic acid binding site. Revertants of the P413L mutations restore the function of EF-G with or without affecting the level of resistance to fusidic acid. The revertant mutations probably restore the balance between the GDP and GTP conformations of EF-G off the ribosome, and most of them are located close to the interface between the G domain and domain II. The procedure for the isolation of pseudo-wild-type forms of EF-G can be used to direct evolution progressively away from the wild-type while still maintaining the essential functions of EF-G.
Johanson
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U
Department of Molecular Biology, Uppsala University Biomedical Centre, Sweden.
Aevarsson
A
A
Liljas
A
A
Hughes
D
D
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005672:Fusidic Acid / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012335:RNA, Ribosomal / Q000235:genetics; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D000198:Spectinomycin / Q000494:pharmacology; D013489:Suppression, Genetic; D016963:Thermus thermophilus / Q000235:genetics
D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011500:Protein Synthesis Inhibitors; D012335:RNA, Ribosomal; D005672:Fusidic Acid; D000198:Spectinomycin