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Promoter sequences after TSS?

cloning transcription mRNA promoter

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#1 doxorubicin

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Posted 19 July 2012 - 05:28 AM

How common is it to find promoter elements within the mRNA sequence? I'm trying to clone the 5'UTR and promoter for a human gene, and the best candidates for promoter elements are actually within the RefSeq mRNA sequence after the transcriptional start site. Is this normal? These same sequences have been demonstrated in mouse to be bona fide promoter elements.

#2 pcrman

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Posted 19 July 2012 - 04:00 PM

What type of promoter elements do you see in the sequence downstream of TSS? Promoter sequence sometimes can extend into the first exon which may contain elements important for transcription. So promoter could be a small region both up and downstream of the TSS.

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Posted 20 July 2012 - 12:50 AM

Thanks pcrman. Maybe I should have just called them transcription factor binding sites. Specifically, I'm referring to the human interferon stimulated gene USP18. In mouse, it has been demonstrated that there are two TF binding sites for the TF IRF2 (Malakhova et al. 2002). Precisely the same sequences exist in human and at least one is predicted to be an interferon stimulated response element (Tsukahara et al. 2006). The only thing is that in mouse, the sites occur prior to the TSS and in human the sites are present within the RefSeq mRNA. I guess I'll just clone the 5'UTR and a couple hundred base pairs prior to it.

Also, do you know how useful this SABiosciences TF binding site prediction tool is?

http://www.sabioscie...ene=n&B2=Search

It also sees an IRF2 site after the TSS, but I think in the wrong position and it tells me there are some other TF binding sites that would make sense for an interferon stimulated gene more than 6000 bps away from the TSS.

#4 pcrman

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Posted 20 July 2012 - 04:51 PM

If it is a TF binding site, it is very possible that one in the adjacent exons or introns, or even far away could regulate transcription. for reporter gene assay, it is also common to include some sequence downstream TSS into the construct upstream of a luciferase gene.

I have never used that program which is hosted by Qiagen and believe it should be a reliable one. But just to make sure you can use another program to do the same prediction to see you get similar results.





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