Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA

被引:58
作者
Janzen, DM
Frolova, L
Geballe, AP
机构
[1] Fred Hutchinson Canc Res Ctr, Div Human Biol, Seattle, WA 98109 USA
[2] Fred Hutchinson Canc Res Ctr, Div Clin Res, Seattle, WA 98109 USA
[3] Univ Washington, Dept Med, Seattle, WA 98115 USA
[4] Univ Washington, Dept Microbiol, Seattle, WA 98115 USA
[5] Russian Acad Sci, VA Engelhardt Mol Biol Inst, Moscow 119991, Russia
关键词
D O I
10.1128/MCB.22.24.8562-8570.2002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 eRFI-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.
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页码:8562 / 8570
页数:9
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