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Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis

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Science  16 Sep 2011:
Vol. 333, Issue 6049, pp. 1630-1632
DOI: 10.1126/science.1208813

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  • RE: Obvious errors with trans-translation assay

    This paper claims to show that POA inhibits trans-translation, but the trans-translation assay that is presented in Fig. 3 is designed in a way that prevents any trans-translation from occurring.
    First, the authors used a template for translation that has 8 AGG codons. A run of rare codons such as this would be likely to lead to trans-translation in vivo, but not in the purified system that is used for the in vitro experiments. In the cell, a run of rare codons causes the ribosome to stall because there are not enough charged cognate tRNAs, and then the mRNA is cut by nucleases resulting in a nonstop ribosome and trans-translation. In the PURExpress kit, all tRNAs are provided in abundance, so there will be no stalling during translation, and there are no ribonucleases so the mRNA will not be cleaved. This template will not lead to trans-translation in vitro.
    Second, the authors did not provide any active tmRNA for the reaction. The tmRNA gene the authors describe transcribing in their methods is the pre-tmRNA that contains 5' and 3' extensions. As for pre-tRNAs, pre-tmRNA cannot be charged and is not active until it is processed by multiple nucleases. These nucleases are not present in the PURExpress kit, so per the authors' description all the tmRNA they added was inactive.
    It is obvious from the gels in Fig. 3 C-E that no significant amount of trans-translation occurred. In vitro trans-translation assays using this method should produce...

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    Competing Interests: I work on trans-translation and have an interest in honest, accurate experiments with this system.

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