Greenwald Emily, Galls Drew, Park Joon, Jain Nimit, Montgomery Stephen B, Roy Bijoyita, Yin Y Whitney, Fire Andrew Z
Department of Genetics, Stanford University, 1291 Welch Road, Stanford, CA 94305, United States.
Department of Pathology, Stanford University, 1291 Welch Road, Stanford, CA 94305, United States.
Nucleic Acids Res. 2025 Mar 20;53(6). doi: 10.1093/nar/gkaf236.
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.
RNA聚合酶（RNAPs）将DNA转录为RNA。包括噬菌体Sp6和T7、大肠杆菌以及小麦胚芽来源的几种RNAPs已被证明能在DNA的3'末端添加核糖核苷酸。线粒体有其自身的RNA聚合酶（mtRNAPs）。通过检测RNAPs作用于具有游离3'末端的DNA分子的反应产物，我们发现酵母和人类mtRNAP制剂表现出用核糖核苷酸延伸DNA 3'末端的强大活性。产生的分子是5'端为输入DNA、3'端为延伸RNA的连续DNA→RNA链。这种链由多种DNA寡核苷酸输入产生，这些寡核苷酸在序列上与DNA 3'末端具有短互补性，RNA部分的序列与输入DNA互补。我们提供了一套基于荧光的检测方法来方便地检测此类产物，并表明这种活性是包括噬菌体RNAPs和多亚基大肠杆菌RNAP在内的多种RNAPs的普遍特性。这些结果支持了一个模型，即DNA既作为引物又作为模板，当DNA的3'末端用核糖核苷酸延长时延伸开始。由于这种DNA→RNA类分子尚未命名，我们提议将其命名为DragonRNA。
