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Enhancer Analysis


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#1 Jab6130

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Posted 25 April 2013 - 01:52 PM

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'
3'TCGCAC5' 3'GTGCGA5'





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.

#2 pcrman

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Posted 25 April 2013 - 02:09 PM

it depends. some TF binding is orientation dependent and some are not.

Take a look at these pages:

http://www.genomatix...AQ_answers.html

[ Transcription factors usually bind in a defined orientation to the DNA double helix. This orientation depends on the orientation of the DNA sequence they recognize, i.e. their transcription factor binding site. An exception are factors that recognize symmetric or palindromic sites. In this case the factor can bind principally in both orientations.

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.[/b]

#3 Jab6130

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Posted 25 April 2013 - 03:59 PM

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?




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