Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli

Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors. Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes. While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking. In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA. An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose. Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E. coli by disrupting transcriptional interference. Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo, despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro. Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.