Fig. 1: 5′ RACE-Seq reveals hyperactive T7 promoter variants.
From: Maximizing transcription of nucleic acids with efficient T7 promoters
a 5′RACE-Seq scheme. A 500 bp dsDNA library harboring a T7 promoter template with randomized nucleotide composition from +2 to +16 (highlighted in red) was transcribed in vitro, using T7 RNA polymerase. The resulting 210 nucleotides long RNAs were reverse transcribed, and the 5′ end of the respective cDNA was converted into a library for deep sequencing. In parallel, an aliquot of the promoter DNA library was directly sequenced to account for potential sequence bias in the template. b Normalized average nucleotide compositions of T7 promoter sequence variants from positions +2 to +16 in amplified RNAs, determined by 5′ RACE-Seq. The region of the extended initiation bubble, which extends from positions −4 to +7, is highlighted in light gray. c Differential promoter activity of +4 to +8 sequence motifs determined by 5′RACE-seq. Shown are the log2 relative abundances of individual sequence motifs. All promoters contain a G at positions +1 to +3. High correlation was observed between two independent experiments. d In vitro transcription reactions comparing +4 to +8 sequence motifs with high, low, and intermediate promoter activity. A 410-nucleotides long RNA was in vitro transcribed for the indicated time points using the displayed promoter variant. Shown is the resulting fold amplification of template DNA. Error bars represent the standard deviation of triplicate experiments.
