Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation.
Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.
Martemyanov
K A
KA
Institute of Protein Research, Russian Academy of Sciences, Pushchino.
Gudkov
A T
AT
D003001:Cloning, Molecular; D004926:Escherichia coli; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion; D016963:Thermus thermophilus / Q000378:metabolism*
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors