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eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1–280) and of eRF3 (positions 1–477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478–530 and 628–637 of eRF3 and positions 281–305 and 411–415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411–415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.
doi:10.1016/j.cellbi.2007.12.005 
Copyright © 2008 International Federation for Cell Biology Published by Elsevier Ltd.
Nuclear localization of eukaryotic class II release factor (eRF3): Implication for the multifunction of eRF3 in ciliates Euplotes cell
Received 30 July 2007;
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Abstract
Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis.
Keywords: Polypeptide release factors; Immunoelectron localization; Nucleus; RNAi interference; Euplotes octocarinatus
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Strains and culture conditions
- 2.2. Antibodies
- 2.3. RNAi and fluorescence observation
- 2.4. Preparation of Euplotes cells, ultrathin sectioning and immunoelectron microscopy
- 3. Results and discussion
- 3.1. Polyclonal antibody
- 3.2. Localization of eRF3 in the cytoplasm
- 3.3. Localization of eRF3 in the macro- and micronucleus
- 3.4. eRF3 gene silencing experiments
- Acknowledgements
- References
