I am working on a eukaryotic gene which is downregulated in pathological conditions at both DNA and protein levels. I studied the 5' region of the gene to find the promoter binding and repressor binding sites using the Ensembl and UCSC browsers. I found that there is no TATA box region in my gene as I couldn't find the respective TATA sequences in the first 600 nucleotides in front of 5' UTR of my gene. I have tried the available public databases as mentioned in one of the answers on ResearchGate, but was not able to find particular proteins using those tools. I found that mass spectrometry analysis is one of the ways to find the DNA binding proteins. What are other advanced methods in finding the DNA binding proteins using in silico and practical methods?
Hi,
you provided too little information on the organism or gene you are woking with.
1) It seems that you are confident about the transcription start site. But are you really sure? Do you really know where your promoter is located?
From you writing this sounds not too convincing.
I think that prior to any in silico analysis you need to be very confident what you are working with. Are you possibly dealing with large introns in the UTRs? Differentially splicing of what you think might be the start? etc...
2) Probabilistc models for binding site analyses work bad on single genes, because of many reasons. So, I suggest, go to the lab and do wet-lab experiments first.
3) What you are talking about is binding protein identification.
Well, as your proteins might possibly bind to many regions of your gene in vitro, you need to be sure about #1)! After that, I still favor yeast one-hybrid assays. Y1H is robust and you don't need t have special equipment or skills. But, it will identify false positives and, hence, you will need to do a lot of extra works to validate your findings. It works best with small target loci (up to 500 bp) or already identified cis-regulatory regions of importance (~20bp).
4) Yes, there are reports that used MS for binding protein identification. This is costly, you need a lot of wet-lab experience and access to respectivce machines. You require a lot of controls to be sure that your binding protein has bound to your probe more often that to suitable mock control probes. AND, you need to be very confident about the region you are interested in.
Otherwise, you might probably miss to answer your scientific question.
hi Dierk, thanks a lot for your valuable suggestion. I am working on rat. As regarding the promoter seqeunce I compared the mRNA of my respective gene to Ensembl and UCSC database and have chosen the first 600 nucleotides infront of 5' UTR . I am not aware of the presence of the introns in that region.
Hi,
just like any mammalian gene, rat genes are spread over large genomic loci. And so are regulatore regions.
Analysing 600bp upstream of the UTR will probably lead to the identification of core-promoter binding proteins.
Important regulatory elements might be located several kb up- or downstream of your gene.
Anyway, 600 bp can be used in yeast one-hybrid screens or in MS based analyses. For MS analyses you will need a lot of clean input material. On the other hand, Y1H will provide only week growth. In both approaches you will need a lot of controls to rule out secondary effects.
Try these tools:
http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3
or:
TRANSFAC
RegulonDB (Procaryotes)
SCPD (yeast)
Muscle-specific regulation of transcription
MatInspector
Yes, the above tools are the ones to go for.
- keep in mind
1) they contain motif consensifor less than 5% of rat/mammalian DNA binding proteins. For full access to e.g. TRANSFAC you need to pay a licence fee.
2) you work with a single gene and , hence, the probabilistic programs e.g. for co-expressed genes, won't work
3) the ones for yeast and bactria will give you fals results and will not work!
Promoters are organized very differently in vertebrates compared with basal eukaryotes or bacteria
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