Maximum distance between ATG & SP6 promoter

Studio401 rupertyip at msn.com
Thu Nov 7 09:36:20 EST 2002


nicholas_theodorakis at urmc.rochester.edu (Nick Theodorakis) wrote in message news:<3dc9aca7.35498706 at netnews.worldnet.att.net>...
> On 6 Nov 2002 14:05:14 -0800, rupertyip at msn.com (Studio401) wrote:
> 
> >I'd like to express a protein with a cDNA that contains ~180 bases
> >upstream of the first ATG.  Will cloning this cDNA into an expression
> >vector (with an SP6 promoter) express protein or is the ATG too far
> >away from the SP6 promoter?
> >
> >Does anyone know if there is a maximum distance between the SP6
> >promoter and the 1st ATG?  Is there are rule of thumb for such things?
> 
> For eukaryotic expression or prokaryotic? In eukaryotes, generally,
> the longer  the 5'utr the greater the tendency to be translated
> poorly. Generally, though, 180 bp is not too unusual. Upstream AUGs
> are also bad for translation, as is too much secondary structure. In
> prokaryotes, I don't think there is any length effect as long as there
> is an appropriate Shine-Dalgarno sequence available at the right
> place.
> 
> Nick


Thanks for your reply Nick

We're trying to in vitro translate a cDNA using Promega's TNT kit. 
Unfortunately, our cDNA was subcloned into a expression vector with
~180 bp in the 5'-UTR upstream of the SP6 promoter.  I was just
wondering if 180 bases was too far away for the SP6 promoter in this
instance for expression.  Are there examples in the literature where
people have expressed proteins where the ATG is =>180 bases away from
the SP6 promoter?



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