Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation.
The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
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Hou
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Yaskowiak
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March
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors