How many nts does T7 polymerase need 5' of the promoter quence to work?

Paul N Hengen pnh at ncifcrf.gov
Mon Oct 27 15:34:56 EST 1997


Dave Adelson (d.adelson at prospect.anprod.csiro.au) wrote:

> Does anyone have at their fingertips just how many nucleotides are
> required 5' of the T7 promoter site for the T7 polymerase to bind and do
> it's thing?  For instance, using a PCR product with the T7 promoter in
> one of the primers.

It depends. There are two issues here: 1) the sequence of DNA where
specific contacts are being made, and 2) the region of DNA being
covered by polymerase.  I think your question is concerned more with
the particlular sequence, and not the length, right? See Figure 3 on
page 6099 of the first reference below and you will see that the
conservation (logo) is quite wide, but you can create a DNA sequence
which would bind T7 polymerase more strongly depending on the
particlular sequence that you would engineer into your PCR primer.
If you select the highest frequency base at each position, you can
get away with less information at the 5' end of the promoter. Be
aware that if you create a VERY strong promoter, you might kill the
cells upon induction. The safe bet is to use a sequence that closely
resembles a naturally occurring T7 promoter. Look at the list in
Figure 9 of the second reference below.

@article{Schneider.Stephens.Logo,
author = "T. D. Schneider
     and R. M. Stephens",
title = "Sequence Logos: A New Way to Display Consensus Sequences",
journal = "Nucl. Acids Res.",
volume = "18",
pages = "6097-6100",
year = "1990"}

@article{Schneider1986,
author = "T. D. Schneider
     and G. D. Stormo
     and L. Gold
     and A. Ehrenfeucht",
title = "Information content of binding sites on nucleotide sequences",
journal = "J. Mol. Biol.",
volume = "188",
pages = "415-431",
year = "1986"}

You also might want to see this paper:

@article{Schneider1989,
author = "T. D. Schneider
     and G. D. Stormo",
title = "Excess Information at Bacteriophage {T7} Genomic Promoters
Detected by a Random Cloning Technique",
journal = "Nucl. Acids Res.",
volume = "17",
pages = "659-674",
year = "1989"}

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