In article <jr.34.000D7D5D at dna.bio.warwick.ac.uk>, jr at dna.bio.warwick.ac.uk writes:
>Dear all,
>> Maybe someone out there can help me with this question regarding the
>T7 RNA promoter. So far as I can find out the promoter sequence is:
>(From the Stratagene catalog)
>>>TAATACGACTCACTATA *GGG
>> in which transcription initiates at the first G residue of the three G's and
>is marked with an asterix
>>My question is : Do those three G's have to be there or could I replace the
>three G's without loss of transcription activity?
>>I would ideally like to replace them with TGC. This is so as to intiate
>transcription at a start of my insert. (Before someone posts asking why I'm
>doing this since translation would occur further down and therefore I
>don't need to change the T7 promoter sequence, let me say its just a long
>story and the work necessitates this change. If you really want to know I
>don't mind E-mailing responses.)
>>>Thanks in advance
>>>Jaz
>According to an article by Milligan et al (Nuc. Acids Res. 15:8783,1987) yields
of transcription product are greatly reduced if the +1 or +2 G are changed
but less affected by changes at +3 base. We have made a construct in our
lab where we changed the +2G to a C with the +1 still G and it works well
in transcription. I would guess that +1 changes would cause big problems
however. Good luck!
Marty Stoltzfus
-
