TF-bindingsite in luciferase - (Sep/12/2006 )

Hi all

I am trying to study the role of a transcription factor and I found that it represses several genes (luciferase assay) but I also found that luciferase has a bindingsite for this transcription factor. What I am worried about is whether the repression I see is due to the effect of binding of my TF to my gene of interest or to binding to luciferase. Does anyone know if it is possible that proteinbinding to the luciferase DNA inhibits its expression?
Im thinking of mutating the bindingsite to be sure, but if anyone has other suggestions, please let me know.

Thanks

just a scientist

-just a scientist-

QUOTE (just a scientist @ Sep 12 2006, 10:59 AM)

Don't you kow about any gene that should be upregulated by your transcription factor? You could first test that by luciferase assay and then you could think about mutating the sequence. But I wouldn't feel like excluding that the binding could lead to the repression of the transcription of the luciferase cDNA. Don't you have a luciferase cloned downstream SV40 or CMV promoter? Maybe that would be enough to see whether there is repression or not.

-dnafactory-

Where is this binding site? In the promoter or the coding region? If it's in the coding region it will probably have minimal effect as it is likely to dissociate when the DNA 'melts' prior to transcription.

I know that Promega do Renilla vectors which have optimised codons to minimise protein binding (the ph-RL series as opposed to the pRL series). Don't know if they do a similar firefly version.

If you mutate the binding site are you sure you'll still get a functional luciferase protein?

-Doc_Martin-