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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

Keyword

Anti-Bacterial Agents

Binding Sites

DNA Mutational Analysis

Directed Molecular Evolution

Drug Resistance, Microbial

Escherichia coli

Fusidic Acid

Models, Molecular

Mutation

Peptide Elongation Factor G

Peptide Elongation Factors

Protein Structure, Tertiary

Protein Synthesis Inhibitors

RNA, Ribosomal

Salmonella typhimurium

Spectinomycin

Suppression, Genetic

Thermus thermophilus

Journal Article

Research Support, Non-U.S. Gov't

Publication and Content Type

art (subject category)

ref (subject category)