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Nucleic Acids Research 2005 33(18):5740-5748; doi:10.1093/nar/gki882
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Published online 6 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast

Pedro A. Ortiz and Terri Goss Kinzy*

Department of Molecular Genetics, Microbiology and Immunology, UMDNJ Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854-5635, USA

*To whom correspondence should be addressed. Tel: +1 732 235 5450; Fax: +1 732 235 5223; Email: kinzytg{at}umdnj.edu

Received July 22, 2005. Revised August 12, 2005. Accepted September 18, 2005.

The eukaryotic translation elongation factor 2 (eEF2), a member of the G-protein superfamily, catalyzes the post-peptidyl transferase translocation of deacylated tRNA and peptidyl tRNA to the ribosomal E- and P-sites. eEF2 is modified by a unique post-translational modification: the conversion of His699 to diphthamide at the tip of domain IV, the region proposed to mimic the anticodon of tRNA. Structural models indicate a hinge is important for conformational changes in eEF2. Mutations of V488 in the hinge region and H699 in the tip of domain IV produce non-functional mutants that when co-expressed with the wild-type eEF2 result in a dominant-negative growth phenotype in the yeast Saccharomyces cerevisiae. This phenotype is linked to reduced levels of the wild-type protein, as total eEF2 levels are unchanged. Changes in the promoter, 5'-untranslated region (5'-UTR) or 3'-UTR of the EFT2 gene encoding eEF2 do not allow overexpression of the protein, showing that eEF2 levels are tightly regulated. The H699K mutant, however, also alters translation phenotypes. The observed regulation suggests that the cell needs an optimum amount of active eEF2 to grow properly. This provides information about a new mechanism by which translation is efficiently maintained.


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