The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants

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The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants. / Johanson, U; Aevarsson, A; Liljas, A; Hughes, D.

In: Journal of Molecular Biology, Vol. 258, No. 3, 10.05.1996, p. 420-32.

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TY - JOUR

T1 - The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants

AU - Johanson, U

AU - Aevarsson, A

AU - Liljas, A

AU - Hughes, D

PY - 1996/5/10

Y1 - 1996/5/10

N2 - 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.

AB - 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.

KW - Anti-Bacterial Agents

KW - Binding Sites

KW - DNA Mutational Analysis

KW - Directed Molecular Evolution

KW - Drug Resistance, Microbial

KW - Escherichia coli

KW - Fusidic Acid

KW - Models, Molecular

KW - Mutation

KW - Peptide Elongation Factor G

KW - Peptide Elongation Factors

KW - Protein Structure, Tertiary

KW - Protein Synthesis Inhibitors

KW - RNA, Ribosomal

KW - Salmonella typhimurium

KW - Spectinomycin

KW - Suppression, Genetic

KW - Thermus thermophilus

KW - Journal Article

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

U2 - 10.1006/jmbi.1996.0259

DO - 10.1006/jmbi.1996.0259

M3 - Article

VL - 258

SP - 420

EP - 432

JO - Journal of Molecular Biology

T2 - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 1089-8638

IS - 3

ER -