By inserting synthetic oligonucleotides into a highly expressed gene in E. coli it has been shown that unfavourable codon usage can reduce the maximum translation rate of a protein. However, in the case of the codon used (AGG), a significant effect on translation was only seen at very high transcription rates from a gene containing multiple copies of the unfavourable codon.
In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter. Analysis of tie pro ei s jroduced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level. A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC. Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled. In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur.
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