In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

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Abstract
To investigate the regulatory mechanism of the tufB operon, we have constructed plasmids in which the lac structural genes have been fused to the regulatory region and the 5′-coding sequence of the tufB gene. The fusion was performed by incorporating the 6.6 kb EcoRI-HpaI fragment of plasmid pTUB1, which carried the tufB gene (Miyajima et al. 1979), into the EcoRI and SmaI sites of pMC1403 lac fusion vector (Casadaban et al. 1980). This gene fusion resulted in the production of a hybrid protein consisting of the N-terminal portion (12 amino acid residues) of EF-TuB and the enzymatically active C-terminal half of β-galactosidase. Bacteria harboring the recombinant plasmid showed a strong Lac+ phenotype. In such a fusion, the lac gene expression was under the control of the tufB promoter. This was evidenced by the following observations; (i) the tufB-lacZ hybrid protein was synthesized constitutively; (ii) its production augmented in parallel with the increase in growth rate; and (iii) on carbon-source upshift, the hybrid protein was produced at a rate 2.5-fold higher than that of the mass increase. Several derivatives of this recombinant plasmid harboring deletions and/or inversions in the tufB regulatory region have been constructed and their properties are described.