PMID:17630323
| Citation |
Spiegel, PC, Ermolenko, DN and Noller, HF (2007) Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome. RNA 13:1473-82 |
|---|---|
| Abstract |
Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP. |
| Links |
PubMed PMC1950763 Online version:10.1261/rna.601507 |
| Keywords |
Escherichia coli; Guanosine Triphosphate/analogs & derivatives; Guanosine Triphosphate/chemistry; Guanosine Triphosphate/metabolism; Hydrolysis; Peptide Elongation Factor G/chemistry; Peptide Elongation Factor G/physiology; Protein Biosynthesis/physiology; Protein Transport; RNA, Bacterial/chemistry; RNA, Bacterial/metabolism; RNA, Messenger/chemistry; RNA, Messenger/metabolism; Ribosomes/chemistry; Ribosomes/metabolism |
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