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.

[1]  S. Krawczyk,et al.  Triple helix formation inhibits transcription elongation in vitro. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Britten,et al.  Gene regulation for higher cells: a theory. , 1969, Science.

[3]  A. Saltzman,et al.  Promoter specificity and modulation of RNA polymerase II transcription , 1989, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  H. Bujard,et al.  lac Repressor blocks transcribing RNA polymerase and terminates transcription. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[5]  E. Goldwasser,et al.  Evidence suggesting negative regulation of the erythropoietin gene by ribonucleoprotein. , 1990, The Journal of biological chemistry.

[6]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[7]  S. Ottonello,et al.  A class III transcription factor composed of RNA. , 1991, Science.

[8]  H. Bujard,et al.  Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. , 1989, Gene.

[9]  J. Majors,et al.  Initiation of in vitro mRNA synthesis from the wild-type lac promoter. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[10]  C. Hélène,et al.  Rational design of sequence-specific oncogene inhibitors based on antisense and antigene oligonucleotides. , 1991, European journal of cancer.

[11]  D. Steege,et al.  Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein. , 1991, Nucleic acids research.

[12]  T. Povsic,et al.  Triple helix formation by oligonucleotides on DNA extended to the physiological pH range , 1989 .

[13]  D. Thomas,et al.  Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes. , 1991, Nucleic acids research.

[14]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[15]  A. Firulli,et al.  Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Adhya,et al.  Multipartite genetic control elements: communication by DNA loop. , 1989, Annual review of genetics.

[17]  S. J. Flint,et al.  Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. Dervan,et al.  Sequence-specific cleavage of double helical DNA by triple helix formation. , 1987, Science.

[19]  D. Steege,et al.  lac repressor blocks in vivo transcription of lac control region DNA. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Miller,et al.  A molecular model for gene repression. , 1966, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Levine,et al.  Transcriptional repression of eukaryotic promoters , 1989, Cell.

[22]  C. Thummel.  Mechanisms of transcriptional timing in Drosophila. , 1992, Science.

[23]  B. Wold,et al.  Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system. , 1992, Biochemistry.

[24]  P. Dervan,et al.  Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Nielsen,et al.  Sequence-selective DNA recognition by synthetic ligands. , 1991, Bioconjugate chemistry.

[26]  R. Burgess,et al.  Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II). , 1987, Biochemistry.

[27]  H. Bujard,et al.  RNA polymerase II transcription blocked by Escherichia coli lac repressor. , 1990, Science.

[28]  Jookyung J Lee,et al.  lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter , 1991, Cell.

[29]  M. Rougée,et al.  Sequence specificity in triple-helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability. , 1991, Biochemistry.

[30]  J. Francois,et al.  Inhibition of restriction endonuclease cleavage via triple helix formation by homopyrimidine oligonucleotides. , 1989, Biochemistry.

[31]  B. Pettitt,et al.  Binding of triple helix forming oligonucleotides to sites in gene promoters. , 1991, Biochemistry.

[32]  B. Wold,et al.  Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation. , 1989, Science.

[33]  D. Praseuth,et al.  Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[34]  B. Wold,et al.  Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA. , 1990, Biochemistry.

[35]  R. Tjian,et al.  Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. , 1989, Science.

[36]  S. J. Flint,et al.  Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. , 1988, Science.

[37]  D. Crothers,et al.  Selective repression of transcription by base sequence specific synthetic polymers. , 1979, Biochemistry.

[38]  F. Birg,et al.  Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent. , 1990, Nucleic acids research.

[39]  L. J. Maher,et al.  Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system. , 1988, Nucleic acids research.

[40]  P. Dervan,et al.  Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation. , 1991, Science.

[41]  F. Studier,et al.  Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. , 1991, Journal of molecular biology.

[42]  R. D. Wells,et al.  Site-specific inhibition of EcoRI restriction/modification enzymes by a DNA triple helix , 1990, Nucleic Acids Res..

[43]  R. Wells,et al.  Specificity of the three-stranded complex formation between double-stranded DNA and single-stranded RNA containing repeating nucleotide sequences. , 1968, Journal of molecular biology.