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Small clusters of divergent amino acids surrounding the effector domain mediate the varied phenotypes of EF‐G and LepA expression
Author(s) -
Yaskowiak Edward S.,
March Paul E.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.tb02363.x
Subject(s) - biology , effector , escherichia coli , amino acid , elongation factor , ef tu , biochemistry , gtpase , microbiology and biotechnology , gene , ribosome , rna
Summary Elongation factors G, Tu, and related proteins (including LepA) form a distinct subgroup within the GTPase superfamily. This observation is based primarily upon amino acid comparisons of the effector region (G2) of the GTP‐binding domain. To examine the functional importance of the highly conserved elongation factor G2 domain a series of chimeric proteins were constructed between Escherichia coli EF‐G and Micrococcus luteus EF‐G, and between E. coli EF‐G and LepA (a protein of unknown function). The M. luteus EF‐G/E. coli EF‐G hybrid, M. luteus EF‐G , and E. coli EF‐G efficiently complemented EF‐G function in an E. coli strain (PEM101) harbouring a temperature‐sensitive mutation in fusA (the gene encoding EF‐G). A comparison of the amino acid sequences of the M. luteus EF‐G and E. coli EF‐G indicated that groups of divergent amino acid residues (amino acids 1–9 and 72–80) were not important for function. LepA and LepA/EF‐G chimeric proteins were tested for the ability to complement EF‐G function in vivo , for cross‐linking to 8‐azido‐[γ‐ 32 ]‐GTP in vitro and for fusidic acid‐dependent co‐sedimentation with 70S ribosomes. With one exception, all chimeras could be readily cross‐linked to azido‐GTP in an EF‐G‐like manner, indicating that hybrid protein construction did not generally result in improperly folded GTP‐binding domains. However, the inability of such chimeras to complement EF‐G function in vivo indicates that the effector domains are not functionally interchangeable. All LepA/EF‐G chimeric proteins were severely defective in fusidic acid‐dependent complex formation with 70S ribosomes. A comparison of the amino acid sequences of all three proteins suggests that residues 30–33, 43–48, and 63–66 of E. coli EF‐G are important for EF‐G specific ribosome‐associated function.