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.

Department of Molecular Genetics of Bacteria Institute of Microbiology Academy of Sciences of the Czech Republic CZ 14220 Prague 4 Czech Republic

Division of Biomolecular Physics Institute of Physics Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 5 121 16 Prague 2 Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Gilead and IOCB Research Center Flemingovo nam 2 CZ 16610 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Gilead and IOCB Research Center Flemingovo nam 2 CZ 16610 Prague 6 Czech Republic Department of Organic Chemistry Faculty of Science Charles University Prague Hlavova 8 CZ 12843 Prague 2 Czech Republic

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Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltrasferases. ChemBioChem. 2002;3:274–293. PubMed

Fu Y., He C. Nucleic acid modifications with epigenetic significance. Curr. Opin. Chem. Biol. 2012;16:516–524. PubMed PMC

Tahiliani M., Koh K.P., Shen Y., Pastor W.A., Bandukwala H., Brudno Y., Agarwal S., Iyer L.M., Liu D.R., Aravind L., et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930–935. PubMed PMC

Kriaucionis S., Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in purkinje neurons and the brain. Science. 2009;324:929–930. PubMed PMC

Ito S., Shen L., Dai Q., Wu S.C., Collins L.B., Swenberg J.A., He C., Zhang Y. Tet proteins can convert 5-methylcytosine to 5- formylcytosine and 5-carboxylcytosine. Science. 2011;333:1300–1303. PubMed PMC

Münzel M., Globisch D., Carell T. 5-Hydroxymethylcytosine, the Sixth Base of the Genome. Angew. Chem. Int. Ed. 2011;50:6460–6468. PubMed

Song C.-X., He C. Potential functional roles of DNA demethylation intermediates. Trends Biochem. Sci. 2013;38:480–484. PubMed PMC

Lu X., Han D., Zhao B.S., Song C.-X., Zhang L.-S., Doré L.C., He C. Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics. Cell Res. 2015;25:386–389. PubMed PMC

Liutkevičiutè Z., Kriukienè E., Ličytè J., Rudytè M., Urbanavičiutè G., Klimašauskas S. Direct decarboxylation of 5-Carboxylcytosine by DNA C5- Methyltransferases. J. Am. Chem. Soc. 2014;136:5884–5887. PubMed

Schiesser S., Pfaffeneder T., Sadeghian K., Hackner B., Steigenberger B., Schröder A.S., Steinbacher J., Kashiwazaki G., Höfner G., Wanner K.T., et al. Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. J. Am. Chem. Soc. 2013;135:14593–14599. PubMed

Bachman M., Uribe-Lewis S., Yang X., Burgess H.E., Iurlaro M., Reik W., Murrell A., Balasubramanian S. 5-Formylcytosine can be a stable DNA modification in mammals. Nat. Chem. Biol. 2015;11:555–557. PubMed PMC

Pfaffeneder T., Spada F., Wagner M., Brandmayr C., Laube S.K., Eisen D., Truss M., Steinbacher J., Hackner B., Kotljarova O., et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat. Chem. Biol. 2014;10:574–581. PubMed

Kass S.U., Pruss D., Wolffe A.P. How does DNA methylation repress transcription? Trends Genet. 1997;13:444–449. PubMed

Law J.A., Jacobsen S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 2010;11:204–220. PubMed PMC

Schröder A., Steinbacher J., Steinberger B., Gnerlich F.A., Schiesser S., Pfaffeneder T., Carell T. Synthesis of a DNA promoter segment containing all four epigenetic nucleosides: 5-Methyl-, 5-Hydroxymethyl-, 5-Formyl-, and 5-Carboxy-2′-deoxycytidine. Angew. Chem. Int. Ed. 2014;53:315–318. PubMed

Lercher L., McDonough M.A., El-Sagheer A.H., Thalhammer A., Kriaucionis S., Brown T., Schofield C.J. Structural insights into how 5-hydroxymethylation influences transcription factor binding. Chem. Commun. 2014;50:1794–1796. PubMed

Wang L., Zhou Y., Xu L., Xiao R., Lu X., Chen L., Chong J., Li H., He C., Fu X.-D., et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex. Nature. 2015;523:621–625. PubMed PMC

Raiber E.-A., Murat P., Chirgadze D.Y., Beraldi D., Luisi B.F., Balasubramanian S. 5-Formylcytosine alters the structure of the DNA double helix. Nat. Struct. Mol. Biol. 2015;22:44–49. PubMed PMC

Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA Methyltransferases. ChemBioChem. 2002;3:274–293. PubMed

Loenen W.A.M., Dryden D.T.F., Raleigh E.A., Wilson G.G., Murray N.E. Nucleic Acids Res. 2014;42:2–19. PubMed PMC

Bickle T.A., Kruger D.H. Biology of DNA Restriction. Microbiol. Rev. 1993;57:434–450. PubMed PMC

Sanchez-Romero M.A., Cota I., Csadesús J. DNA methylation in bacteria: from the methyl group ot the methylome. Curr. Opin. Microbiol. 2015;25:9–16. PubMed

Reisenauer A., Shapiro L. DNA methylation affects the cell cycle transcription of the CtrA global regulator in Caulobacter. EMBO J. 2002;21:4969–4977. PubMed PMC

Seo Y.J., Matsuda S., Romesberg F.E. Transcription of an expanded genetic alphabet. J. Am. Chem. Soc. 2009;131:5046–5047. PubMed PMC

Ishizuka T., Kimoto M., Sato A., Hirao I. Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry. Chem. Commun. 2012;48:10835–10837. PubMed

Liu J., Doetsch P. W. Escherichia coli RNA and DNA polymerase bypass of dihydrouracil: mutagenic potential via transcription and replication. Nucleic Acids Res. 1998;26:1707–1712. PubMed PMC

You C., Wang J., Dai X., Wang Y. Transcriptional inhibition and mutagenesis induced by N-nitroso compound-derived carboxymethylated thymidine adducts in DNA. Nucleic Acids Res. 2015;43:1012–1018. PubMed PMC

Viswanathan A., Doetsch P. W. Effects of nonbulky DNA base damages on Escherichia coli RNA polymerase-mediated elongation and promoter clearance. J. Biol. Chem. 1998;273:21276–21281. PubMed

Kuraoka I., Endou M., Yamaguchi Y., Wada T., Handa H., Tanaka K. Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription-coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem. 2003;278:7294–7299. PubMed

Farnham P.J., Platt T. Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1982;79:998–1002. PubMed PMC

Kitsera N., Stathis D., Lühnsdorf B., Müller H., Carell T., Epe B., Khobta A. 8-Oxo-7, 8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1. Nucleic Acids Res. 2011;39:5926–5934. PubMed PMC

You C., Wang Y. Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo. Nat. Protoc. 2015;10:1389–1406. PubMed PMC

Macíčková-Cahová H., Pohl R., Hocek M. Cleavage of functionalized DNA containing 5-modified pyrimidines by Type II restriction endonucleases. ChemBioChem. 2011;12:431–438. PubMed

Mačková M., Pohl R., Hocek M. Polymerase synthesis of DNA bearing vinyl groups in major groove and their cleavage by restriction endonucleases. ChemBioChem. 2014;15:2306–2312. PubMed

Mačková M., Boháčová S., Perlíková P., Poštová Slavětínská L., Hocek M. Polymerase synthesis and restriction enzyme cleavage of DNA containing 7-substituted 7-deazaguanines. ChemBioChem. 2015;16:2225–2236. PubMed

Macíčková-Cahová H., Hocek M. Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases. Nucleic Acids Res. 2009;37:7612–7622. PubMed PMC

Kielkowski P., Brock N.L., Dickschat J.S., Hocek M. Nucleobase protection strategy for gene cloning and expression. ChemBioChem. 2013;14:801–804. PubMed

Kielkowski P., Macíčková-Cahová H., Pohl R., Hocek M. Transient and switchable (triethylsilyl)ethynyl protection of DNA against cleavage by restriction endonucleases. Angew. Chem. Int. Ed. 2011;50:8727–8730. PubMed

Vaníková Z., Hocek M. Polymerase synthesis of photocaged DNA resistant against cleavage by restriction endonucleases. Angew. Chem. Int. Ed. 2014;53:6734–6737. PubMed

Seela F., Thomas H. Synthesis of certain 5-Substituted 2′-deoxytubercidin derivatives. Helv. Chim. Acta. 1994;77:897–903.

Krásný L., Gourse R.L. An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J. 2004;23:4473–4483. PubMed PMC

Anthony L.C., Artsimovitch I., Svetlov V., Landick R., Burgess R.R. Rapid purification of His(6)-tagged Bacillus subtilis core RNA polymerase. Protein Expr. Purif. 2000;19:350–354. PubMed

Chang B.Y., Doi R.H. Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor. J. Bacteriol. 1990;172:3257–3263. PubMed PMC

Wiedermannová J., Sudzinová P., Kovaľ T., Rabatinová A., Šanderová H., Ramaniuk O., Rittich Š., Dohnálek J., Zhihui F., Halada P., et al. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis. Nucleic Acids Res. 2014;42:5151–5163. PubMed PMC

Rabatinová A., Šanderová H., Jirát Matějčková J., Korelusová J., Sojka L., Barvík I., Papoušková V., Sklenář V., Žídek L., Krásný L. The δ subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell. J. Bacteriol. 2013;195:2603–2611. PubMed PMC

Hocek M., Fojta M. Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids. Org. Biomol. Chem. 2008;6:2233–2241. PubMed

Hollenstein M. Nucleoside triphosphates - building blocks for the modification of nucleic acids. Molecules. 2012;17:13569–13591. PubMed PMC

Hocek M. Synthesis of base-modified 2′-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology. J. Org. Chem. 2014;79:9914–9921. PubMed

Ludwig J. A new route to nucleoside 5′-triphosphates. ActaBiochim. Biophys. Acad. Sci. Hung. 1981;16:131–133. PubMed

Kovacs T., Otvos L. Simple synthesis of 5-vinyl and 5-ethynyl-2′-deoxyuridine-5′-triphosphates. Tetrahedron Lett. 1988;29:4525–4528.

Ruff E.F., Drennan A.C., Capp M.W., Poulos M.A., Artsimovitch I., Record M.T., Jr E. coli RNA polymerase determinants of open complex lifetime and structure. J. Mol. Biol. 2015;427:2435–2450. PubMed PMC

Bralley P., Chang S.A., Jones G.H. A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNAnucleotidyltransferases. J. Bacteriol. 2005;187:5927–5936. PubMed PMC

Murakami K.S. Structural biology of bacterial RNA polymerase. Biomolecules. 2015;5:848–862. PubMed PMC

Weiss A., Shaw L.N. Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria. FEMS Microbiol. Rev. 2015;39:541–554. PubMed PMC

Ruff E.F., Record M.T., Jr, Artsimovitch I. Initial events in bacterial transcription initiation. Biomolecules. 2015;5:1035–1062. PubMed PMC

Sojka L., Kouba T., Barvík I., Sanderová H., Maderová Z., Jonák J., Krásny L. Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis. Nucleic Acids Res. 2011;39:4598–4611. PubMed PMC

Fukushima T., Ishikawa S., Yamamoto H., Ogasawara N., Sekiguchi J. Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis. J. Biochem. 2003;133:475–483. PubMed

Lei Y., Oshima T., Ogasawara N., Ishikawa S. Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis. J. Bacteriol. 2013;195:1697–1705. PubMed PMC

Ménová P., Dziuba D., Güixens-Gallardo P., Jurkiewicz P., Hof M., Hocek M. Fluorescence quenching in oligonucleotides containing 7-substituted 7-deazaguanine bases prepared by the Nicking Enzyme Amplification Reaction. Bioconjugate Chem. 2015;26:361–366. PubMed

Krásný L., Tišerová H., Jonák J., Rejman D., Šanderová H. The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis. Mol. Microbiol. 2008;69:42–54. PubMed

Lane W.J., Darst S.A. Molecular evolution of multisubunit RNA polymerases: sequence analysis. J. Mol. Biol. 2010;395:671–685. PubMed PMC

Lane W.J., Darst S.A. Molecular evolution of multisubunit RNA polymerases: structural analysis. J. Mol. Biol. 2010;395:686–704. PubMed PMC

Whipple F.W., Sonenshein A.L. Mechanism of initiation of transcription by Bacillus subtilis RNA polymerase at several promoters. J. Mol. Biol. 1992;223:399–414. PubMed

Liu X., Bushnell D.A., Kornberg R.D. Lock and key to transcription: sigma-DNA Interaction. Cell. 2011;147:1218–1219. PubMed

Feklistov A., Darst S.A. Structural basis for promoter -10 element recognition by the bacterial RNA polymerase sigma subunit. Cell. 2011;147:1257–1269. PubMed PMC

Zuo Y., Steitz T.A. Crystal structures of the E. coli transcription initiation complexes with a complete bubble. Mol. Cell. 2015;58:534–540. PubMed PMC

Bae B., Feklistov A., Lass-Napiorkowska A., Landick R., Darst S.A. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife. 2015;4:e08504. PubMed PMC

Campbell E.A., Muzzin O., Chlenov M., Sun J.L., Olson C.A., Weinman O., Trester-Zedlitz M.L., Darst S.A. Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol. Cell. 2002;9:527–539. PubMed

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