Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 2715-2722 |
| Number of pages | 8 |
| Journal | Nucleic Acids Research |
| Volume | 26 |
| Issue number | 11 |
| DOIs | |
| State | Published - Jun 1 1998 |
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All Science Journal Classification (ASJC) codes
- Genetics
Cite this
Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli. / Soukup, Garrett; Maher, L. James.
In: Nucleic Acids Research, Vol. 26, No. 11, 01.06.1998, p. 2715-2722.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli
AU - Soukup, Garrett
AU - Maher, L. James
PY - 1998/6/1
Y1 - 1998/6/1
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/26.11.2715
DO - 10.1093/nar/26.11.2715
M3 - Article
VL - 26
SP - 2715
EP - 2722
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 11
ER -