Inhibition of T7 RNA polymerase initiation by triple-helical DNA complexes: a model for artificial gene repression.
An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides. This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation. Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner. Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected. Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions. Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter. The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes. In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation.
Maher
L J
LJ
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases