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The T7 polymerase doesn't transcribe all sequences equally well, the transciption efficiency can vary widely for different sequences. One well known requirement of the T7 polymerase is that the sequence should begin with 2-3 guanines for efficient transcription.

Is there any literature that quantified the effects of the sequence on the transcription efficiency? I'm mostly interested in the effect of the first few nucleotides, as changing other parts of the sequence is usually not possible.

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  • $\begingroup$ Would be nice to know why some of my T7-promoter plasmids produce ~100 mg protein/L of culture and others maybe 2-3. $\endgroup$
    – Nick T
    Commented Dec 15, 2011 at 13:29
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    $\begingroup$ Well, the protein expression also depends on the strength of the ribosome binding site. $\endgroup$
    – Gergana Vandova
    Commented Dec 16, 2011 at 19:00
  • $\begingroup$ I'm doing in vitro transcriptions and I'm not translating the RNA anyway, so for me that part doesn't matter $\endgroup$
    – Mad Scientist
    Commented Dec 16, 2011 at 19:16
  • $\begingroup$ This is not an answer, but a starting point. I found this article Bai L, Santangelo TJ, Wang MD. Single-molecule analysis of RNA polymerase transcription. Annu Rev Biophys Biomol Struct. 2006;35:343-60. that may contain the answer to your question. Lacking that, you may find good starting points in the references section. $\endgroup$
    – KAM
    Commented Dec 26, 2011 at 12:54

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By far the most important part is the very beginning of the transcript, especially the positions +1 and +2. The conservered consensus sequence in class III T7 promoters is GGGAGA, any changes in the first two nucleotides severely reduce the transcription efficiency. Changes at positions +3 to +6 have much smaller effects.

Additionally, changes that put many AU base pairs in that region (e.g. GGUUU) seem to affect the trancription efficiency negatively.

Other sequences that are problematic anywhere, not only in the beginning are long stretches of uridines or adenines. Sequences with eight or more uridines or adenines can cause the polymerase to slip, which results in transcripts with more uridines or adenines than in the template.

These characteristics are detailed in the paper from Milligan and Uhlenbeck from 1989.

Milligan, J. F. & Uhlenbeck, O. C. Synthesis of small RNAs using T7 RNA polymerase. Meth. Enzymol. 180, 51–62 (1989).

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