lambda bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA.
Expression of the bacteriophage lambda 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 beta-lactamase-encoding gene of the vector as a reporter. The results show that expression of bar+ minigene severely inhibits beta-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.
Hernández-Sánchez
J
J
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740 Mexico City, Mexico.
Valadez
J G
JG
Herrera
J V
JV
Ontiveros
C
C
Guarneros
G
G
D010582:Bacteriophage lambda / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002474:Cell-Free System; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis*; D012355:RNA, Transfer, Ile / Q000096:biosynthesis*; D012357:RNA, Transfer, Lys / Q000096:biosynthesis*
D012346:RNA, Transfer, Amino Acyl; D012355:RNA, Transfer, Ile; D012357:RNA, Transfer, Lys; C019037:tRNA, peptidyl-; D012343:RNA, Transfer; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase