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. 2014 May;20(5):609–620. doi: 10.1261/rna.042523.113

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© 2014 Koutmou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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FIGURE 3. — Pelleting assay measuring the binding of RF3-nucleotide to different ribosome complexes. (A) Empty 70S ribosomes pelleted following incubation with RF3 bound to GDP, GTP, and GDPNP. Only RF3:GDPNP is detectable after pelleting, suggesting that RF3:GTP can interact with ribosomes in the absence of RF1. (B) Termination complexes formed with His-RF1 were pelleted after incubation with RF3 bound to GDP, GTP, and GDPNP in the presence and absence of an unlabeled RF1 chase. These results indicate that RF3:GDPNP and RF3:GTP can promote RF1 dissociation from the ribosome. In all experiments, RF1 and RF3 are His-tagged and their presence detected by Western blot.