Effect of carcinogenic adducts on transcription by T7 RNA polymerase

The effect of modifying the T7 promoter-containing plasmid pDR100 with aminofluorene (AF), acetylaminofluorene (AAF), and benzo[a]pyrene (B[a]P) adducts on RNA synthesis by the T7 RNA polymerase was determined. We found that increasing levels of each of the three adducts caused a progressive decline in RNA synthesis, but that the inhibition produced by benzo[a]pyrene adducts was substantially greater than that caused by either AF or AAF adducts. Polyacrylamide gel electrophoresis analysis confirmed that the B[a]P adducts more strongly inhibited chain elongation since their presence resulted in the production of RNA fragments having average chain lengths very close to that predicted if each adduct permanently blocked the polymerase during transcription. We have also determined the effect of each type of adduct on the initiation of transcription and show that the T7 RNA polymerase initiates as efficiently on both AF or AAF-containing templates as it does on unmodified DNA, while B[a]P adducts were found to increase in the number of chain starts.