DragonRNA: Generality of DNA-primed RNA-extension activities by DNA-directed RNA polymerases.

Abstract

RNA polymerases (RNAPs) transcribe DNA into RNA. Several RNAPs, including from bacteriophages Sp6 and T7, Escherichia coli, and wheat germ, had been shown to add ribonucleotides to DNA 3' ends. Mitochondria have their own RNAPs (mtRNAPs). Examining reaction products of RNAPs acting on DNA molecules with free 3' ends, we found yeast and human mtRNAP preparations exhibit a robust activity of extending DNA 3' ends with ribonucleotides. The resulting molecules are serial DNA→RNA chains with the input DNA on the 5' end and extended RNA on the 3' end. Such chains were produced from a wide variety of DNA oligonucleotide inputs with short complementarity in the sequence to the DNA 3' end with the sequence of the RNA portion complementary to the input DNA. We provide a set of fluorescence-based assays for facile detection of such products and show that this activity is a general property of diverse RNAPs, including phage RNAPs and multi-subunit E. coli RNAP. These results support a model in which DNA serves as both primer and template, with extension beginning when the 3' end of the DNA is elongated with a ribonucleotide. As this DNA→RNA class of molecule remains unnamed, we propose the name DragonRNA.