Fig. 2.

RNA ligase-activity of the recombinant Pyrobaculum RtcB protein. Suitable tRNA splicing intermediates (see Materials and Methods) were incubated with the Pyrobaculum RtcB—MBP fusion protein. Lane A: no ATP or GTP added. Lane B: no ATP or GTP but with a heavy metal mix (39). Lanes C–F: the enzyme preparation was preincubated in 0.5 mM ZnCl₂ for 15 min on ice before the enzyme was added to the RNA ligation mixture. Lane C: no ATP or GTP added; lane D: with 0.5 mM ATP; lane E: with 0.5 mM GTP; lane F: with 0.5 mM ATP and 0.5 mM GTP. Lane G: no MBP-RtcB enzyme added. Lane H: the positive control—addition of T4 polynucleotide kinase with 3′-phosphatase (PNKp) and T4 RNA ligase 1. Lanes D–H include NTPs that serve as coprecipitant during the ethanol precipitation of the phenol/chloroform extracted RNA ligation mixtures. Hence, more ribonucleic acids are precipitated as indicated by the presence of more tRNA halves in lanes D–H in comparison to lanes A–C.