T7 transcription in yeast
dbaggott at reed.edu
Thu Jan 11 15:15:25 EST 1996
rdlab at gandalf.bio.uci.edu (rlab davis) wrote:
>In article <lagilb-090196143517 at yyqi1.monsanto.com> lagilb at ccmail.monsanto.com (Larry Gilbertson) writes:
>>Has anyone ever expressed T7 RNA polymerase in yeast (Saccharomyces) and
>>used it to turn on a gene with a T7 promoter?
>Chen, W., S. Tabor, and K. Struhl. 1987. Distinguishing between mechanisms
>of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase.
>Cell 50: 1047-1055.
>They use the T7 promoter + RNAP to drive his3 in yeast.
>Martin Hoyt (R H Davis lab)
>Mol. Biol. and Biochem.
>Univ. of California, Irvine
>mahoyt at uci.edu
I would hasten to add that in the Chen et al. paper, although they did get transcription of HIS3 from the T7 promoter in a T7 RNA pol. dependent manner, transcription did not rescue his3 mutants (i.e. no functional his3 protein). The authors suggest that the his3 transcripts were not translated because the transcripts were not capped.
For a paper dealing directly with use of this system in yeast see Benton et al. (MCB 10: 353-360). They used a modified T7 RNA pol. with the SV40 nuclear localization signal and got import of the pol. into the nucleus as well as specific transcription from T7 promoters. Again however, they did not get translation of their target genes possibly because of secondary structure at the 5'-end of the transcripts. (The same transcripts were not translated in an in vitro system unless a 5' hairpin structure was removed.) They did not do localization studies to see whether the transcripts were making it out of the nucleus so that could also be a problem.
There is also a paper (Pinkham et al., MCB 14: 4643-4652) in which T7 RNA pol is equipped with a mitochondrial localizing signal and used to transcribe a gene from a T7 promoter (integrated into the mitochondrial genome) and the transcripts ARE translated. So it seems there is nothing intrinsic (eg no 5' cap) to transcription by T7 RNA pol. that makes translation impossible.
I wonder if the absence of functional proteins in the other papers is a result of the inability of the T7-derived transcripts to make it out of the nucleus. Although there is at least one paper using a mammalian system in which T7-derived transcripts from nuclear plasmids are translated (sorry, I can't track down the reference off hand).
If by chance anyone knows anything more about this I would love to hear...
Reed College Biology Dept.
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