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Nucleic Acids Research Advance Access originally published online on June 25, 2008
Nucleic Acids Research 2008 36(13):4233-4241; doi:10.1093/nar/gkn395
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Nucleic Acids Research, 2008, Vol. 36, No. 13 4233-4241
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Eva Jacinto-Loeza, Serafín Vivanco-Domínguez, Gabriel Guarneros and Javier Hernández-Sánchez*

Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México D. F., 07000, México

*To whom correspondence should be addressed. Tel: +(52-55) 5061 3800 ext 5352; Fax: +(52-55) 5747 7100; Email: javierh{at}cinvestav.mx

Received March 14, 2008. Revised June 5, 2008. Accepted June 5, 2008.

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 µg/µl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.


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