Small clusters of divergent amino acids surrounding the effector domain mediate the varied phenotypes of EF-G and LepA expression.
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-[gamma-32P]-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.
Yaskowiak
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ES
School of Microbiology and Immunology, University of New South Wales, Sydney, Australia.
March
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PE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005672:Fusidic Acid / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D005816:Genetic Complementation Test; D006160:Guanosine Triphosphate / Q000378:metabolism; D016982:Micrococcus luteus / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu