Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations.
A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.
Mesters
J R
JR
Department of Biochemistry, Leiden University, The Netherlands.
Potapov
A P
AP
de Graaf
J M
JM
Kraal
B
B
D000494:Allosteric Regulation / Q000502:physiology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004586:Electrophoresis; D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000187:drug effects / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D011728:Pyridones / Q000494:pharmacology; D012270:Ribosomes / Q000378:metabolism*; D013307:Streptomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011728:Pyridones; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; C008536:mocimycin; D013307:Streptomycin