Targeted 3′–5′ polymerase activity on RNA duplexes catalyzed by Thg1/TLP enzymes. (A) Schematic of the reaction catalyzed with RNA duplexes, in which a radioactively labeled 5′-triphosphorylated 26-mer RNA substrate transcript (S) is annealed to a 5′-hydroxylated (unlabeled) RNA template oligonucleotide (T). The annealed duplexes contain an 8-nt 3′-overhang (5′-GCGCGCGC-3′) to serve as a template for 3′–5′ polymerase activity of the tested Thg1/TLP enzymes. Template-dependent 3′–5′ addition of either CTP or GTP (depending on the templating nucleotide, as shown) will result in incorporation of nucleotides into the 5′-end of the labeled S, and these elongated products are visualized by denaturing gel electrophoresis. (B) A survey of purified Thg1/TLP enzyme activities with the 19 bp RNA duplex, as shown in A. Reactions were performed as described in Materials and Methods, with individual enzymes indicated under each lane (Sc: ScThg1, Hs: HsThg1, A2: AcaTLP2, D4: DdiTLP4, Ms: MsTLP, Mx: MxTLP, and Bt: BtTLP). The A2-G lane corresponds to a reaction where GTP has been omitted, and CTP is the only nucleotide added to the reaction. The last four lanes contain purified catalytically inactive Thg1/TLP variants, as indicated. The first two lanes of the gel are no enzyme controls indicating the position of the unreacted 26-mer substrate (lane S), and of the unreacted duplex (lane S + T), which demonstrates that migration of the substrate is not affected by the presence of the template RNA on this denaturing (20% polyacrylamide/8 M urea) gel. The thick black line marks the position of the unreacted substrate, and bands corresponding to nucleotide addition products are labeled accordingly. (C) A survey of purified Thg1/TLP enzyme activities with the 8 bp RNA duplex, performed as described in Materials and Methods. Individual enzymes were indicated as described above, with the addition of D3: DdiTLP3 for this assay.
