Transcription Factor??? - ID (Apr/30/2010 )
Hi Guys! 
So I'm wondering if anyone knows if there's a 'final say' assay on what is or is not a transcription factor?
Basically, I'm trying to design a project and have a protein I THINK is a transcriptional regulator, because it's homolog is a txn factor, and some of the proteins it binds to are also txn regulators. I'm already planning to do a microarray to look at gene expression, followed by EMSA to see if it binds DNA in promoter regions of any genes that I get from the microarray.
If I have candidate genes, is the luciferase reporter assay the commonly used experiment to definitely say 'this is a txn factor'?
I'm not familiar with the luciferase assay. I gather that I put the promoter region of a candidate gene in front of the luciferase gene. When I transfect it in, with a plasmid for my protein, I look to see if transcript levels change? What if my protein requires co-activators?
Any input would be awesome. Thanks a bunch!!
Hi, you can consult with the following papers. To check if your protein has transcription activation activity, you can try cotransfecting your protein in the presence of your candidate promoter hooked to a reporter gene. Also, since you already have a candidate gene/promoter region that you think it would bind to, you can do a CHIP assay to confirm the presence of your protein binding to DNA. In the case of your protein requiring other coactivators to enhance transcription, you may need to cotrasfect the other coactivators in the reporter assays.
Good luck!!
Science. 1999 Jun 25;284(5423):2174-7.
Regulation of transcription by a protein methyltransferase.
Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, Aswad DW, Stallcup MR.
CoCoA, a nuclear receptor coactivator which acts through an N-terminal activation domain of p160 coactivators.
Kim JH, Li H, Stallcup MR.
Mol Cell. 2003 Dec;12(6):1537-49.
Mol Cell. 2008 Aug 22;31(4):510-9.
CCAR1, a key regulator of mediator complex recruitment to nuclear receptor transcription complexes.
Kim JH, Yang CK, Heo K, Roeder RG, An W, Stallcup MR.
aye on Apr 30 2010, 04:33 PM said:
So I'm wondering if anyone knows if there's a 'final say' assay on what is or is not a transcription factor?Basically, I'm trying to design a project and have a protein I THINK is a transcriptional regulator, because it's homolog is a txn factor, and some of the proteins it binds to are also txn regulators. I'm already planning to do a microarray to look at gene expression, followed by EMSA to see if it binds DNA in promoter regions of any genes that I get from the microarray.
If I have candidate genes, is the luciferase reporter assay the commonly used experiment to definitely say 'this is a txn factor'?
I'm not familiar with the luciferase assay. I gather that I put the promoter region of a candidate gene in front of the luciferase gene. When I transfect it in, with a plasmid for my protein, I look to see if transcript levels change? What if my protein requires co-activators?
Any input would be awesome. Thanks a bunch!!