Oligoribonucleotide-based gene-specific transcription inhibitors that target the open complex.
We have demonstrated that oligoribonucleotides that lack a 3'-OH group and cannot be extended by RNA polymerase can hybridize to the single-stranded DNA formed inside the transcription initiation bubble (or open complex) and inhibit transcription. Using the lacUV5/Escherichia coli RNA polymerase or trpEDCBA/E. coli RNA polymerase transcription system as a model, we have found that effective inhibitors are five nucleotides in length and must be complementary to the DNA template strand in the region from -5 to +2 about the transcription start site (designated +1). We have used the DNA cleavage activity of 1,10-phenanthroline-copper to confirm that the mechanism of inhibition is via oligoribonucleotide hybridization to the open complex and have used this cleavage chemistry to demonstrate that these oligonucleotide inhibitors hybridize in an antiparallel orientation to their DNA target. Systematic modification of the parent phosphodiester oligoribonucleotide pentamer revealed that the phosphorothioate backbone-containing analogs have increased open complex binding affinity and are more effective transcription inhibitors than their phosphodiester counterparts.
Milne
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L
Department of Biological Chemistry, School of Medicine, and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1570, USA.
Perrin
D M
DM
Sigman
D S
DS
D001483:Base Sequence; D003300:Copper / Q000737:chemistry; D004247:DNA / Q000187:drug effects*; D018076:DNA, Complementary / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005821:Genetic Techniques; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000737:chemistry; D009843:Oligoribonucleotides / Q000737:chemistry*; D010618:Phenanthrolines / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*
D018076:DNA, Complementary; D009696:Nucleic Acids; D009843:Oligoribonucleotides; D010618:Phenanthrolines; D003300:Copper; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C025205:1,10-phenanthroline