The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.
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.
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics*; D012984:Software
D012333:RNA, Messenger; D007371:Interferon-gamma; D004247:DNA; D004262:DNA Restriction Enzymes