Nagpal, N.; Tai, A. K.; Fong, Y. W.; Agarwal, S.

RNA synthesis by eukaryotic polymerases requires existing polynucleotides to serve as templates or primers. Here, we describe primer- and template-free RNA generation by human terminal nucleotidyltransferase 4B (TENT4B) via de novo polymerization of free nucleotides. We observed that recombinant TENT4B (rTENT4B) consumes ATP to yield inorganic pyrophosphate in the absence of a primer or template, concurrent with the appearance of oligomeric poly-adenosine RNA products. Remarkably, 5' labels on gamma-phosphate-modified ATP or GTP are retained during polymerization in the presence of unlabeled nucleotide triphosphates (NTPs). These polymers are created at a similar efficiency irrespective of the inclusion of a primer, indicating robust RNA synthesis by rTENT4B from free NTPs. While canonical purine NTPs are favored, nucleotide diphosphates can also serve as substrates for rTENT4B-mediated de novo RNA polymerization. rTENT4B-mediated RNA synthesis using free adenosine nucleotides shows high processivity to generate 1000s-mers, whereas guanosine nucleotide polymerization is strongly and uniformly self-limited and yields a 3'-exonuclease-resistant oligonucleotide. Interrogation of other RNA polymerases reveals potential capacity for de novo polymerization using free ATP, albeit at significantly higher substrate concentrations and lower efficiency compared to rTENT4B. Our data provide definitive evidence of efficient template-free de novo RNA synthesis by a eukaryotic polymerase.