Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates

Abstract

Synthesis of T7 RNA polymerase is inhibited by the presence of bulky adducts in the DNA template. Of the types of adducts tested, those formed by the potent carcinogen benzo[a]pyrene (B[a]P) caused the greatest inhibition. M13 DNA molecules containing a single late T7 RNA polymerase promoter have been prepared containing B[a]P adducts in either the displaced or template strand and these have been used as templates for in vitro RNA transcription by the T7 RNA polymerase. We find that the level of inhibition of RNA synthesis is substantially greater (greater than or equal to 10-fold) when adducts are positioned specifically in the template strand. Polyacrylamide gel analysis of the products synthesized off these strand-specifically modified templates showed that adducts situated in the template strand gave rise to discrete bands which presumably represent the termination of synthesis at the adduct site while the product derived from a template containing adducts in the displaced resembled that obtained using a native template.