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. 1984 Oct 25;12(20):7663–7675. doi: 10.1093/nar/12.20.7663

The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.

L H Tessier, P Sondermeyer, T Faure, D Dreyer, A Benavente, D Villeval, M Courtney, J P Lecocq
PMCID: PMC320192  PMID: 6093047

Abstract

Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.

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Selected References

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