Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein

We attempted to devise a transcription system in which a particular DNA seq uence of interest could be inducibly expressed under the control of a modif ied polymerase ill (pol III) promoter. Its activation requires a mutated tr anscription factor not contained endogenously in human cells. We constructe d such a promoter by fusing elements of the beta -lactamase gene of Escheri chia coil, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene. This construct functionally resembl es a 5'-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence. Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt), Its transcription the refore requires TBP-DR2 exclusively instead of TBPwt, In order to render th e system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells, induction of such a stable and viable clone with tetracycline resulted in t he expression of functional TBP-DR2. This system may conceptually be used i n the future to inducibly express an arbitrary DNA sequence in vivo under t he control of the above mentioned promoter.