| The goal of this study was to investigate effects of protein overexpression on the physiology of Escherichia coli. Effects of protein overexpression can be divided into two aspects, the general effect common to all protein overexpression, and the specific effects particular to the biochemical activity of the protein overexpressed.;The general effect was investigated by overexpressing an inactive protein, LuxA (the alpha subunit of luciferase). Results led to the following model. When a gene is overexpressed, its elevated transcripts bind to a large number of ribosomes. The rest of the mRNA pool has to compete for the reduced number of free ribosomes. Therefore, some of the genes involved in essential metabolism are down-regulated, leading to reduced growth rate. The reduced growth rate is then sensed by the growth rate control mechanism, which reduces the rate of rRNA synthesis. Protein overexpression also leads to the destruction of ribosomes (18). The reduced rate of rRNA synthesis and increased degradation rate result in a reduced ribosome number, which further intensifies the competition for free ribosomes.;The effect of phosphoenolpyruvate carboxykinase (Pck) overexpression was characterized as an example of the specific effect of protein production. Pck overexpression retards growth more than the overexpressional non-functional protein, LuxA, possibly because the increased enzymatic activity perturbs the metabolite pools, which in turn upset global regulation. In particular, we found that Pck overexpression abolishes the induction of glnA transcription under nitrogen limitation in a strain deficient in phosphotransacetylase, which is responsible for the reversible inter-conversion of acetyl coenzyme A and acetyl phosphate. This effect was not observed when a Pck mutant, Pck51, which has a new oxaloacetate decarboxylase activity, was overexpressed. Therefore, the specific effect of overexpressing Pck is attributable to the PEP-formation activity of Pck. |
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