Specific inhibition of transcription by triple helix-forming oligonucleotides.
Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.
Duval-Valentin
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G
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, Institut National de la Santé et de la Recherche Médicale Unité 201, Centre National de la Recherche Scientifique, Paris, France.
Thuong
N T
NT
Hélène
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C
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D011564:Furocoumarins / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D001618:beta-Lactamases / Q000235:genetics
D011564:Furocoumarins; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D001618:beta-Lactamases