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. 2018 Dec 22;47(3):1428–1439. doi: 10.1093/nar/gky1275

Figure 1.

Figure 1.

Chaetomium Trl1 ligase activity. (A) Cartoon depiction of the domain organization of full-length (FL) 846-aa C. thermophilum Trl1 consisting of N-terminal ligase (LIG), central kinase (KIN), and C-terminal cyclic phosphodiesterase (CPD) domains. An N-terminal 407-aa segment is shown here to comprise an autonomous LIG module. (B) Aliquots (10 μg) of the Superdex fractions of recombinant Trl1-FL and Trl1-LIG were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (C) RNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 2 mM DTT, 10 mM MgCl2, 100 μM GTP, 100 μM ATP, 0.5 pmol (50 nM) 32P-labeled 10-mer HORNA2′p (shown at the bottom with the 32P-label indicated by •), and either no enzyme (lane –), 1.25 μg plant tRNA ligase (AtRNL), or 0.625, 1.25, 2.5, 5, 10 or 25 ng Trl1-FL (from left to right in the titration series) were incubated at 37°C for 30 min. The labeled RNAs were resolved by urea-PAGE and visualized by autoradiography. The positions of the 5′-OH, 2′-PO4 RNA substrate, 5′-PO4, 2′-PO4 kinase reaction product, and the 10-mer circle product of intramolecular ligation are indicated on the left. (D) Reactions mixtures (10 μl) containing 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 2 mM DTT, 10 mM MgCl2, 100 μM GTP, 100 μM ATP, 0.5 pmol (50 nM) 32P-labeled 10-mer HORNA2′p, 250 ng AtKIN-CPD (where indicated by +), and 5 or 10 pg Trl1-LIG (where indicted by + and ++, respectively) were incubated at 37°C for 30 min. The labeled RNAs were resolved by urea-PAGE and visualized by autoradiography.