Triple helix formation inhibits transcription elongation in vitro

Abstract

We have identified a 15-nucleotide site within a G-free transcription cassette that forms triple helix with sequence-specific oligodeoxyribonucleotides. When oligodeoxynucleotides were added to template DNA prior to in vitro transcription, a significant fraction of transcripts were truncated at a site corresponding to the region of triple helix formation. Kinetic analysis of the transcription products demonstrated that these truncated transcripts could be elongated to full length upon prolonged incubation. When an alkylating base was incorporated into the oligodeoxynucleotide to form covalent triple helix, most of the transcripts remained truncated. We conclude that triple helix formation can stall or, in the case of covalent crosslinking, can block RNA polymerase II and thus may provide a method for the specific inhibition of gene expression.