DNA binding ( GO:0003677 ), RNA binding ( GO: 0003723), RNA-directed DNA polymerase activity ( GO: 0003964)
To improve expression
The syngag was designed on the basic of the Pr55gag amino acid sequence employing a codon
occurring most frequently in highly expressed mammalian genes. By this procedure, almost
wooble position within the wild-type coding region was changed to a G or C, resulting in a
nucleotide composition and decreased AT content.
Kofman, A.; Graf, M.; Deml, L.; Wolf, H.; Wagner, R.
Institute for Medical Microbiology and Hygiene, University of Regensburg, D-93053 Regensburg, Germany.
Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation
Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.
Tsitologiia. 45(1): 94-100.
To determine whether codon usage differences affect exclusively translational efficiency of mammalian genes products, wild type gag (wtgag) and synthetic gag (syngag) genes were used in the experiments. Synthetic gene was designed on the basic of the Pr55gag amino acid sequence employing a codon usage occurring most frequently in highly expressed mammalian genes. By this procedure, almost every wooble position within the wild-type coding region was changed to a G or C, resulting in a diverse nucleotide composition and decreased AT content. In order to study the influence of codon usage optimization on gag expression, chimeric constructs were also designed by substituting wtgag fragments with part of syngag mRNA. For nuclear expression, as expected, wtgag expression was extremely low, while the expression of cyngag was the highest among all the constructs leading to the counclusion that HIV1 gag expression depends on the mRNA nucleotide content. However, for the cytoplasmic expressions, all wtgag, syngag, and the chimeric constructs efficiently expressed.