Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

We produced K2Orf3p as N-terminal His-and GST-tagged proteins using an E. coli T7-based system or as an N-terminal His-tagged fusion protein using a baculovirus expression system. We purified the His-and GST-tagged K2Orf3 proteins to homogeneity using appropriate affinity chromatography and tested them for GTase and MTase activities. We readily detected GTase activity of the purified recombinant K2Orf3 proteins in all cases (data not shown); however, their MTase activity was not detected (data not shown). These results are in good agreement with the previously described results of Tiggemann and co-workers (17), who had shown that K2Orf3p possesses TPase and GTase activities in vitro but not MTase activity. They explained this result by using a C-terminal His-tag for the heterologous production of the K2Orf3p capping enzyme that may have interfered with the C-terminal MTase domain (17). We produced the putative K2Orf3p capping enzyme N-terminally fused with either His or GST tags but also failed to detect MTase activity using several in vitro approaches.