TY - JOUR
T1 - Repression of bacteriophage promoters by DNA and RNA oligonucleotides
AU - Skoog, John U.
AU - James maher, L.
N1 - Funding Information:
We acknowledge the excellent technical assistance of D.Eicher, C.Mountjoy, and W.Olivas. We thank W.Gmeiner, B.Gold, and M.Hollingsworth for comments on the manuscript. This work was supported, in part, by a Young Investigator's Award from Abbott Laboratories, and by Grant 5 P30 CA36727-O8 from the National Cancer Institute.
PY - 1993/5/11
Y1 - 1993/5/11
N2 - 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 trlple-hellx formation at two kinds of homopurlne operator sequences [Maher, L. J., Ill, (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 pyrlmldlne 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 trlple-helix Inhibition constant (Ki) estimates of approximately 2 × 10-7 M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators {K, = 6 × 10-7 M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fall 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.
AB - 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 trlple-hellx formation at two kinds of homopurlne operator sequences [Maher, L. J., Ill, (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 pyrlmldlne 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 trlple-helix Inhibition constant (Ki) estimates of approximately 2 × 10-7 M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators {K, = 6 × 10-7 M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fall 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.
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U2 - 10.1093/nar/21.9.2131
DO - 10.1093/nar/21.9.2131
M3 - Article
C2 - 8502553
AN - SCOPUS:0027231901
SN - 0305-1048
VL - 21
SP - 2131
EP - 2138
JO - Nucleic acids research
JF - Nucleic acids research
IS - 9
ER -