Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.