The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form.
Translocation of the tRNA x mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4 degrees C, 20 degrees C, and 37 degrees C. The binding affinity of EF-G is higher to GDP than to GTP at 4 degrees C, but lower at 37 degrees C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the gamma-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an "activity chimera," with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its "ratcheted state," with hybrid tRNA binding sites.
Department of Cell and Molecular Biology, Molecular Biology Program, Uppsala University, S-75 124 Uppsala, Sweden.
Vasili
V
Hauryliuk
Vladimir A
VA
Mitkevich
Natalia A
NA
Eliseeva
Irina Yu
IY
Petrushanko
Måns
M
Ehrenberg
Alexander A
AA
Makarov
D001419:Bacteria / Q000235:genetics; D002151:Calorimetry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D046911:Macromolecular Substances; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism*; D013816:Thermodynamics
D046911:Macromolecular Substances; D020653:Peptide Elongation Factor G; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate