inclusion of translation start site when cloning promoter insert - (Sep/27/2006 )

Hi,

I'm a newbie in doing promoter experiments. Does one include the translation start site as part of the promoter insert to be cloned into a vector or does one just clone the region upstream from the start of the mRNA.. If not, what is reason for excluding the translation start site ? If the region in my gene has multiple transcription initiation sites how does that affect how I do the promoter assays?

thank u

I agree, you do not need to include the ATG start codon but should include you transcription start sites.

As for multiple sites affecting your assays; I guess it depends which assays you are running. If you are worried check the literature again to see if one particular site is favoured or if there are conditions that will cause this.

Additionally you might be able to mutate the other sites and leave just one (that you consider to be the most important).

You need to include the -10 and -30 TATA boxes, although there may be other regulatory elements upstream so cloning 200-300 bp should be good. Also, there is the Shine-Delgarno sequence about 6 bp upstream of the ATG star codon that the ribosome recognises to begin translation. So you should include that too..basically clone everything upsteam of the ATG start codon.

Hi,

I'm a newbie in doing promoter experiments. Does one include the translation start site as part of the promoter insert to be cloned into a vector or does one just clone the region upstream from the start of the mRNA.. If not, what is reason for excluding the translation start site ? If the region in my gene has multiple transcription initiation sites how does that affect how I do the promoter assays?

thank u

thanks a lot!