Repression of bacteriophage promoters by DNA and RNA oligonucleotides.
We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids rather than polypeptides. An in vitro model system involving repression of bacteriophage T7 RNA polymerase initiation has been employed to demonstrate that certain DNA oligonucleotides can repress transcription by site-specific triple-helix formation at two kinds of homopurine operator sequences [Maher, L. J., III, (1992) Biochemistry 31, 7587-7594]. Recognition in the purine motif is based on antiparallel oligonucleotide binding (G.G.C and T.A.T triplets). Recognition in the pyrimidine motif is based on parallel oligonucleotide binding (C+.G.C and T.A.T base triplets). Using this system, we report that the concentration-dependence of repression by DNA oligonucleotides provides triple-helix inhibition constant (Ki) estimates of approximately 2 x 10(-7) M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators (Ki = 6 x 10(-7) M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fail to bind the purine motif operator. Partial substitution of deoxyribose residues tends to rescue repressor activity by RNA oligonucleotides in the purine motif. These results suggest prospects for, and constraints on, natural and artificial RNA-based repressors.
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
J U
JU
Skoog
L J
LJ
Maher
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000187:drug effects; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D009843:Oligoribonucleotides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*; D014764:Viral Proteins
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases