I was wondering if somebody has ever tried to inhibit transcription for a while and then replace the medium, with no inhibitor, in order to see the transcriptions being recovered.
Which drug have you used?
My recollection is that alpha-amanitin inhibition is irreversible because it triggers degradation of Rpb1, the largest RNAP II subunit. However I don't know if the same thing happens using Actinomycin D or DRB or something else.
I can suggest to use DNA damaging agents: Cisplatin or 1,10-Phenanthroline. DNA-damage induces arrest of transcription and activated DNA repair mechanisms. After removing of DNA-damaging agent, DNA in repaired and transcription is going on.
I would suggest DRB which reversibly inhibits the transition from initiation to elongation by blocking phosphorylation of the CTD of RNA pol II. Elongation is not inhibited though so transcription will be ongoing until RNA polymerases has cleared all genes. Good luck!
We are using Actinomycin D in yeast. It is easy to remove and to make cells grow in fresh medium. I know that Alfa-amanitin works also very well
Flavopiridol, roscovitine I have heard work well to inhibit transcription but I have not used them personally.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology