It really depends on the cis-regulating elements that control your promoter. Some promoters extend only over ~50bp (eg mazEF) while some other extend to >500bp (ie csgD, rpoS)

Nathan Fraikin

Thank you very much. I really appreciate the specific examples.

Do you think the promoters are likely to extend through upstream genes (intragenic)? I notice csgD promoter, although very large, does not go through any upstream genes -- there is a very large intergenic region.

I think it is pretty uncommon (but not unknown) that a promoter would overlap an upstream gene. Also pay attention to whether your gene might be in an operon.

Michael J. Benedik

Thank you very much. Experimentally, if I wish to capture natural promoters for recombinant expression, am I fairly safe in assuming the promoter lies in the intergenic region, and will not extend into the upstream gene?

(Of course it happens in some cases, but I ask if it's quite rare.)

Currently, I struggle a lot with capturing the sequence for natural promoters. If I take too little, I could lose regulatory elements. If take too much, I capture portions of the upstream sequence, which may get translated and have some dominant negative effects on the protein encoded by the upstream gene.

I think if the intergenic region is >50 nts then you are correct. However I would not worry about capturing some upstream sequence, the dominant negative effect of inadvertent translation is exceedingly rare with a partial gene.

Michael J. Benedik

Thank you again. I very much appreciate your help.

I do worry that in my case, dominant negative effects may be more common, although I would very much value your thoughts and opinions:

In making genetic knockouts, I replace a "target gene" with an antibiotic resistance cassette.

But I do not want to affect the downstream gene's promoter.

If the downstream gene's promoter sequence is within my "target gene," I would then have to to leave the 3' sequence of my "target gene," and only delete the 5' end of my "target gene."

However, the antibiotic cassette has a strong promoter and very strong transcription. The 3' end of my "target gene" may get co-transcribed very strongly from this cassette promoter, increasing the odds of dominant negative affect severely.

Do you think this fear of dominant negative effects is valid in this case? Or are the odds very low?

Amy Johnson

Thank you, Amy. While I'm primarily concerned about bacteria, I would appreciate hearing the answer in the context of yeast -- likely I can still glean something.