>One relates to your statement:After allowing the recombinant AT1-R mRNA to reach a steady-state, its transcription can be selectively suppressed by adding anhydrotetracycline (Antet). This approach reveals the instrinsic decay rate of the mRNA and avoids toxicities associated with the use of general transcriptional inhibitors such as actinomycin D, which might interfere with normal AT1-R mRNA regulation and many other cellular processes.
>How does Antet inhibit transcription? It will be useful if you can guide me to a good reference on the mechanism of action, effectiveness of inhibition and selectivity of this agent.
The promoter is activated by the tTA protein, a fusion between a tetracycline response element binding protein and the VP16 transactivation domain. The tetracycline response elements are placed upstream of the minimal promoter (either minimal CMV or minimal AT1-R gene promoters have been used). AnTetracycline is thought to bind to the tTA and inhibits binding to the DNA elements. Without a transactivator, the promoters are fairly weak.
I was probably inaccurate in stating the drug inhibits transcription; suppression by relief of transactivation would be more accurate.
Selectivity? In our experimental design, we can measure endogenous AT1-R transcripts simultaneously. We see no affect of Antet on this, so with respect to the native transcript, the drug is highly selective.
The reference is: Gossen, M., and H. Bujard. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc.Natl.Acad.Sci. 89: 5547-5551, 1992.
>The second relates to the stability of mRNA. I am impressed that you have PKA-dependent system. What mRNA domains are involved - coding region or anything in the UTR also?
We are still conducting some experiments along the lines of identifying if a cis-acting mRNA element is necessary for the response to PKA. We have not ruled out that this reflects some general affect on global stability and translation in the preparation. I wish I could say more, but it would be premature at this time.