Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators

Abstract

A rat insulin gene which was fused to Escherichia coli signals for the initiation of translation could not be retained when expressed from the strong rrnB ribosomal RNA promoters or an induced trp/lac (= tac) hybrid promoter. When the latter promoter was repressed by transformation into a lac-repressor-overproducing strain, the insulin gene fragment could be retained. Upon induction of the promoter with isopropyl-beta-D-galactosidase the growth rate of the cells was reduced, and in most cases the cells subsequently lysed. Deletion of the translational initiation signals, changing the reading frame, or insertion of an efficient transcription terminator between the promoter and the rat insulin gene each permitted retention of the fragment. The first two observations indicate that overproduction of the specific polypeptide, and not of the RNA, is detrimental to the cell. The third finding has been exploited for the testing and selection of transcription terminators. The rpoC terminator, which is located distal to the rplJL /rpoBC operon, has been shown to terminate transcripts from the rrnB promoters. It was also shown that the putative rrnB terminators, T1 and T2, each function separately in vivo.