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Nucleic Acids Research, 2003, Vol. 31, No. 20 5949-5956
© 2003 Oxford University Press

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Zemfira N. Karamysheva1,2, Andrey L. Karamyshev2, Koichi Ito2, Takashi Yokogawa3, Kazuya Nishikawa3, Yoshikazu Nakamura2 and Senya Matsufuji*,1

1 Department of Biochemistry II, The Jikei University, School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan, 2 Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8630, Japan and 3 Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

*To whom correspondence should be addressed. Tel: +81 3 3433 1111; Fax: +81 3 3436 3897; Email: senya{at}jikei.ac.jp
Present addresses:
Zemfira N. Karamysheva, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA
Andrey L. Karamyshev, Department of Medical Biochemistry and Genetics, College of Medicine, Texas A&M University System, Health Science Center, College Station, TX 77843-1114, USA

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.


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I. P. Ivanov and J. F. Atkins
Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation
Nucleic Acids Res., March 19, 2007; 35(6): 1842 - 1858.
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