Enhancer Analysis - (Apr/25/2013 )
I am doing an enhancer analysis for a particular transcription factor. I have poured over the literature for an answer to this question, but to no avail. Often transcription factor binding sites are given 5'-->3' for example 5'AGCGTG3'. Which means that this transcription factor will bind sequences 5'AGCGTG3' However I have found that orientation is not important and that the sequence 5'CACGCT3'
will also facilitate binding. However my question is, will 3'AGCGTG5' also act as a binding site? or is strictly 5'-->3' prime direction important? In other words will the following sequences, 3' AGCGTG 5' or its reverse orientation 3'CACGCT 5'
5'TCGCAC 3' 5' GTGCGA 3'
also allow for binding? I have been stumped on this facet for quite some time, and the project is dependent on identifying such sequences before I can start wet lab mutagenesis. Any help would be greatly appreciated.
it depends. some TF binding is orientation dependent and some are not.
Take a look at these pages:
Some transcription factor binding sites must have a defined orientation relative to the promoter or the transcription start site, an example is the TATA-box. Most transcription factor binding sites can occur in both orientations in promoters or enhancers.
Therefore, for the TATA-box only the (+)-strand matches should be considered as true positives (if the strand orientation of the promoter sequence analyzed is known and is in 5'-->3' orientation relative to the gene). For most other transcription factor binding sites both, (+)- and (-)-strand matches should be considered equally.
In addition, there is a technical aspect that has to be considered. Transcription factor binding sites are represented by weight matrices (or IUPAC strings). Each matrix has a strand orientation which depends on the strand orientation of its training sequences used. Therefore, a matrix match on the (+)-strand only means that the matching sequence has the same strand orientation relative to the training sequences used for the matrix generation (and vice versa).
http://www.ncbi.nlm....43696 Opposite orientations of a transcription factor heterodimer bind DNA cooperatively with interaction partners but have different effects on interferon-b gene transcription.
Thanks for the reply PCRman, I've seen that page and that paper. They do a great job to show that TF can often bind for example 5'AGCGTG3' on both the + and - strand (different orientation). How I understand it however this is not the same as saying it can bind 3'AGCGTG5', see what I mean? Anyone have any insight?