Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome.
Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.
Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia.
K A
KA
Martemyanov
A T
AT
Gudkov
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005672:Fusidic Acid / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011691:Puromycin / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D016963:Thermus thermophilus / Q000378:metabolism*
D001426:Bacterial Proteins; D020653:Peptide Elongation Factor G; D006153:Guanosine Diphosphate; D011691:Puromycin; D005672:Fusidic Acid; D006160:Guanosine Triphosphate