Everything on Promotors

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Monique

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Does anyone know how far upstream a promotor can go from a gene in higher eukaryotes??

It is very difficult to define a promotor (which contains regulatory elements for a gene), since it is all non-coding DNA with lots of 'junk' in it..

It is fairly easy to determine the transcription start site (TSS) at the 5'UTR, you just isolate mRNA's and look how far upstream they go, and that way determine the major initiation site..

anything upstream of that would be promotor and I guess that the regulatory elements with the most impact would be directly flanking this TSS?
 

Another God

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ummm....I am thinking several kilobases, but I am not entirely sure.

uh huh: Alberts, on page 400 says that they are known to be up to 50kb away.

I know that the identification of promoters is usually done be nested deletions. DO you know about them? They get the gene and its upstream/downstream region and cut out small sections of it, starting way upstream, working their way down. For each section they measure its rate of transcription. This way they identifiy what sections influence the transcription rate etc.

What is the 5'UTR? I've never heard of it. And how do you 'look upstream' from the mRNA's? Oh, do you mean that once u have the mRNA's, you can line the mRNA code up with the DNA code and then count 35 upstream on the DNA and find the TATA box etc...?

Regulatory elements can actually exist downstream and within the gene as well as upstream.
 

Monique

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Originally posted by Another God
ummm....I am thinking several kilobases, but I am not entirely sure.

uh huh: Alberts, on page 400 says that they are known to be up to 50kb away.
Whoohoo! That is a long bit to analyze, isn't it?? [:P] right now I am focussing on a 500 bp bit and there are hundreds of transcription factor binding sites!

I know that the identification of promoters is usually done be nested deletions. DO you know about them? They get the gene and its upstream/downstream region and cut out small sections of it, starting way upstream, working their way down. For each section they measure its rate of transcription. This way they identifiy what sections influence the transcription rate etc.
That is correct, you can transform a cell line with pieces of promotor (of different sizes) and determine which piece of promotor is able to restore the original level of transcription (by luciferase assay for instance). The tricky part is that there are enhancers and repressors binding the the noncoding DNA, how do you take those into account if they are lying on different part of the promotor?

What is the 5'UTR? I've never heard of it.
That is the 5'Untranslated Region, transcription of a gene starts with an ATG right? That is the start codon, the place of the transcription start site. That DOESN'T mean that the protein start there, though. During translation the enzyme complex will bind to this 5' region, translation start usually 30 bp downstream. The 5'UTR is thus conserved after splicing, it is noncoding DNA, which usually contains sites for enhancers or repressors.

And how do you 'look upstream' from the mRNA's? Oh, do you mean that once u have the mRNA's, you can line the mRNA code up with the DNA code and then count 35 upstream on the DNA and find the TATA box etc...?
Well, you can look at a protein and determine the introns and exons on the DNA right? But how do you determine where the TSS (transcription start site) is, where the promotor ends and the 5'UTR starts? You isolate mRNA and you sequence them in 3'to 5'direction, where the sequence ends, that is where the TSS is.

Regulatory elements can actually exist downstream and within the gene as well as upstream.
Yes, I've also heard those can be in introns, but mainly upstream, since that is where the enzyme complexes bind.
 

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