λ bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA

Expression of the bacteriophage λ two‐codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl‐tRNA hydrolase (Pth). It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl‐tRNA (p‐tR...

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Published in:The EMBO journal 1998-07, Vol.17 (13), p.3758-3765
Main Authors: Hernández-Sánchez, Javier, Valadez, Juan Gerardo, Vega Herrera, Jesús, Ontiveros, Carlos, Guarneros, Gabriel
Format: Article
Language:English
Subjects:
Bacteriophage lambda - genetics
Carboxylic Ester Hydrolases - metabolism
Cell-Free System
Gene Expression Regulation, Viral
Genes, Viral
in vitro minigene expression
peptidyl-tRNA accumulation
peptidyl-tRNA hydrolase
Protein Biosynthesis
protein-synthesis inhibition
RNA, Transfer - biosynthesis
RNA, Transfer, Amino Acyl - biosynthesis
RNA, Transfer, Ile - biosynthesis
RNA, Transfer, Lys - biosynthesis
tRNA starvation
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Summary:Expression of the bacteriophage λ two‐codon, AUG AUA, barI minigene (bar+) leads to the arrest of protein synthesis in cells defective in peptidyl‐tRNA hydrolase (Pth). It has been hypothesized that translation of the bar+ transcript provokes premature release and accumulation of peptidyl‐tRNA (p‐tRNA). Inhibition of protein synthesis would then result from either starvation of sequestered tRNA or from toxicity of accumulated p‐tRNA. To test this hypothesis and to investigate the cause of arrest, we used a coupled in vitro transcription–translation system primed with DNA containing bar+ and the β‐lactamase‐encoding gene of the vector as a reporter. The results show that expression of bar+ minigene severely inhibits β‐lactamase polypeptide synthesis by Pth‐defective extracts and partially inhibits synthesis by wild‐type extracts. Fractions enriched for Pth, or a homogeneous preparation of Pth, prevented and reversed bar+‐mediated inhibition. A mutant minigene, barA702, which changes the second codon AUA (Ile) to AAA (Lys), was also toxic for Pth‐defective cells. Expression of barA702 inhibited in vitro polypeptide synthesis by Pth‐defective extracts and, as with bar+, exogenous Pth prevented inhibition. Addition of pure tRNALys prevented inhibition by barA702 but not by bar+. Expression of bar+ and barA702 led to release and accumulation of p‐tRNAIle and p‐tRNALys respectively but bar+ also induced accumulation of p‐tRNALys. Finally, bar+ stimulated association of methionine with ribosomes probably as fMet‐tRNAfMet and the accumulation of methionine and isoleucine in solution as peptidyl‐tRNA (p‐tRNA). These results indicate that minigene‐mediated inhibition of protein synthesis involves premature release of p‐tRNA, misincorporation of amino acyl‐tRNA, accumulation of p‐tRNAs and possibly sequestration of tRNAs.
ISSN:0261-4189
1460-2075
1460-2075
DOI:10.1093/emboj/17.13.3758