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Molecular and Cellular Biology, December 2002, p. 8562-8570, Vol. 22, No. 24
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.24.8562-8570.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

Deanna M. Janzen,1, Lyudmila Frolova,1,2, and Adam P. Geballe1,3*

Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109,1 Departments of Medicine and Microbiology, University of Washington, Seattle, Washington 98115,2 Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, Moscow 119991, Russia3

Received 31 May 2002/ Returned for modification 8 July 2002/ Accepted 19 September 2002

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.

* Corresponding author. Mailing address: Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Mailstop C2-023, P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-5122. Fax: (206) 667-6523. E-mail: ageballe{at}fhcrc.org.

Molecular and Cellular Biology, December 2002, p. 8562-8570, Vol. 22, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.24.8562-8570.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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