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Promoter strength driving TetR determines the regulatory properties of Tet-controlled expression systems.

ABSTRACT: Bacteria frequently rely on transcription repressors and activators to alter gene expression patterns in response to changes in the surrounding environment. Tet repressor (TetR) is a paradigm transcription factor that senses the environmental state by binding small molecule effectors, the tetracyclines. However, recently isolated peptides that act as inducers of TetR after having been fused to the C-terminus of a carrier protein, suggest that TetR can also regulate gene expression in a signal-transduction pathway. For this shift in regulatory mechanism to be successful, induction of TetR must be sensitive enough to respond to an inducing protein expressed at its endogenous level. To determine this regulatory parameter, a synthetic Tet-regulated system was introduced into the human pathogen Salmonella enterica serovar Typhimurium and tested for inducibility by a peptide. Reporter gene expression was detected if the peptide-containing carrier protein Thioredoxin 1 was strongly overproduced, but not if it was expressed at a level similar to the physiological level of Thioredoxin 1. This was attributed to high steady-state amounts of TetR which was expressed by the promoter of the chloramphenicol acetyl transferase gene (P(cat)). Reducing P(cat) strength either by directed or by random mutagenesis of its -10 element concomitantly reduced the intracellular amounts of TetR. Sensitive and quantitative induction of TetR by an inducing peptide, when it was fused to Thioredoxin 1 at its native locus in the genome, was only obtained with weak P(cat) promoter variants containing GC-rich -10 elements. A second important observation was that reducing the TetR steady-state level did not impair repression. This permits flexible adjustment of an inducible system's sensitivity simply by altering the expression level of the transcription factor. These two new layers of expression control will improve the quality and, thus, the applicability of the Tet and other regulatory systems.

PROVIDER: S-EPMC3407185 | BioStudies |

REPOSITORIES: biostudies

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