Transcription of left-handed Z-DNA templates: increased rate of single-step addition reactions catalyzed by wheat germ RNA polymerase II.
Wheat germ RNA polymerase II is able to transcribe polynucleotide templates in the poly-[d(G-C)] family, adopting either the right-handed B or left-handed Z conformations depending on the ionic environment and temperature. Thus, with poly[d(G-C)] either the B state (in MgCl2) or the associated Z* state (in MnCl2) can be established. Poly[d(G-m5C)] adopts the Z form readily in MgCl2, and poly-[d(G-br5C)] can be regarded as being "constitutively" in the Z state. In transcription studies with CpG as a primer and templates in the left-handed conformation, it is found that the rate of productive elongation, i.e., the synthesis of poly[r(G-C)], is depressed, in accordance with the results of previous studies. However, with a single triphosphate substrate, CTP, the rate of formation of the first phosphodiester bond, i.e., the synthesis of CpGpC, is about 4-fold greater with both the Z and Z* templates than with B-DNA. This transcriptional activity is also catalytic in the sense that product concentrations exceed that of the enzyme. The synthesis of CpGpC is reduced in the presence of GTP. However, the apparent Km value for GTP utilization is lower for the trinucleotide synthesis (0.1 microM) than that obtained for productive elongation (0.8 microM), a result that also holds for B-DNA templates. All transcription reactions are specifically inhibited by the fungal toxin alpha-amanitin, and, in the case of the left-handed templates, by monoclonal anti-Z-DNA antibodies. The relative probabilities of single-step addition and productive elongation imply that the major distinction between transcription of templates in the B and Z conformations involves a step following the synthesis of the first phosphodiester bond. As a result, fully competent elongation complexes do not form on the left-handed DNA.
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Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
Marmillot
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Jovin
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D002712:Chlorides; D004247:DNA / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D009690:Nucleic Acid Conformation; D010944:Plants / Q000201:enzymology*; D011089:Polydeoxyribonucleotides; D012319:RNA Polymerase II / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014908:Triticum / Q000201:enzymology
D002712:Chlorides; D017895:Manganese Compounds; D011089:Polydeoxyribonucleotides; D015636:Magnesium Chloride; D008345:Manganese; D004247:DNA; D012319:RNA Polymerase II; D008274:Magnesium; C025340:manganese chloride