A comparison of the wild type MthRnl and the MthRnl mutants in step 3 of the ligation reaction with various acceptors. Enzymes were assayed using three different acceptors with identical sequence: RNA (RNA30), 2’-O-methylated at 3’-end RNA (RNA30-OMe) or ssDNA (DNA30), and the donor AppDNA17-NH2 as described in the Methods. (A) The control reactions without enzyme. (B) The reactions with non-adenylated form of the wild type MthRnl. (C, D and E) The reactions with K97A, K246A and K246Q MthRnl mutants respectively. The products were separated on 15% urea-PAGE and visualized with SYBR Gold stain. Positions of substrates, ligated products and by-product pDNA17-NH2 are indicated on the right. Acceptor substrates used in the reactions are indicated on the top. Mr indicates molecular weight RNA markers.