PCR-based construction of promoter/G-free templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts.

In vitro transcription has been used for dissecting transcriptional controls in many eukaryotic systems. One modification which greatly reduces background non-specific transcription is the placement of a guanosine-free (G-free) region of DNA immediately downstream from a promoter [Sawadogo and Roeder, Proc. Natl. Acad. Sci. USA 82 (1985) 4394-4398]; transcription in the presence of RNase T1 and 3' O-Me-GTP eliminates non-specific transcripts, and produces the G-free transcripts initiated at the promoter. Restriction site-based fusion of a G-free cassette downstream from promoters is complicated by the requirement for G nucleotides to be excluded from the coding strand downstream from the transcription start points. We present an approach to add a G-free template onto a eukaryotic promoter by combining PCR-based termini construction and terminal deoxynucleotidyl transferase extension. The pisatin demethylase promoter (PDA1p) of the filamentous fungus Nectria haematococca was used as the test promoter. Three PDA1p/G- free constructs were tested in heterologous Drosophila melanogaster and HeLa and homologous N. haematococca transcription extracts. Each extract produced a PDA1p-specific transcript from each construct, but the relative level of transcription between constructs varied, particularly in the homologous extract. Since the choice of G-free sequence influences transcription differently among systems, this method for producing multiple G-free constructs should be useful for constructing and selecting optimal promoter/G-free templates for in vitro transcription in other homologous systems.