Posted 03 March 2009 - 10:57 PM
I have found TESS and TFSEARCH but would like any other recommendations for free websites to may help.
I am also having trouble interpreting the results from these sites.
To me it makes sense that only transcription/receptor sites on the sense/top strand would be able to activate genes. Is this correct? And does that mean I can ignore all found potential binding site indicated as being on the (-) strand?
I am very confused and overwhelmed by the number of potential binding sites that I seem to have!
Posted 04 March 2009 - 07:26 AM
Use the program to give you guide of what is potentially there and then you will have to prove it experimentally.
Sort of deflating, but that's what molecular biology is all about
Posted 04 March 2009 - 04:32 PM
I would look up the consensus for you protein and analyze the sequence by eye.
When looking for binding sites make deletions of your promoter and see what deletions
affect the transcription when your transfect your transcription factor into the cells.
When you narrow the region down to ~200 base pairs you can do mutagenesis and EMSA.
Posted 05 March 2009 - 03:01 PM
Have found my promoter sequence has about 90% homology with an already described promoter region in a closely related species. So this has been helpful!
I have another question too, the fragment that I obtained by Genome Walking in 5kb, pretty big compared to promoter regions of other species. If there anyway to predict the actual promoter size, ie is it possible to have cloned beyond to start of the promoter? Most other published promoters for this gene are around 2kb.
Appreciate your help,
Posted 05 March 2009 - 07:49 PM
Clone the 5 Kb region, put it into a luciferase, transfect in your transcription factor, do deletions and
see what region is necessary for response to your factor.