Which sequence characteristics influence the transcription efficiency of T7 polymerase?
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. &amp; Uhlenbeck, O. C. Synthesis of small RNAs using T7 RNA polymerase. Meth. Enzymol. 180, 51–62 (1989).