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Nucleic Acids Research, 1984, Vol. 12, No. 20 7663-7675
© 1984

Articles

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

Luc-Henri Tessier, Paul Sondermeyer, Thérèse Faure, Dominique Dreyer, Annie Benavente, Dominique Villeval, Michael Courtney and Jean-Pierre Lecocq

Transgene S.A., 11 Rue de Molsheim, 67000 Strasbourg, France

Received August 31, 1984. Accepted October 2, 1984.

Parameters influencing the efficiency of expression of the human ininune interferon (IFW-{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 n 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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