title: Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.
abstract: DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Raindlová
authors.affiliation: Department of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Janoušková
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Slavíčková
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Pavla
authors.initials: P
authors.lastname: Perlíková
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Soňa
authors.initials: S
authors.lastname: Boháčová
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Nemanja
authors.initials: N
authors.lastname: Milisavljevič
authors.affiliation: Department of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Šanderová
authors.affiliation: Department of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Benda
authors.affiliation: Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, 121 16 Prague 2, Czech Republic.
authors.forename: Ivan
authors.initials: I
authors.lastname: Barvík
authors.affiliation: Department of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-14220 Prague 4, Czech Republic krasny@biomed.cas.cz.
authors.forename: Libor
authors.initials: L
authors.lastname: Krásný
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic hocek@uochb.cas.cz.
authors.forename: Michal
authors.initials: M
authors.lastname: Hocek
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D003854:Deoxyribonucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases