Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression ofOrf3p and proof of guanylyltransferase and mRNA-triphosphatase activities

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity c hromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a Functional protein in a bacterial s ystem, The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities, When the lysine residue present at pos ition 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an impo rtant role for the transfer of GRIP from CTP to the 5'-diphosphate end of t he mRNA. Our in vitro data provides the first direct evidence that the poly peptide encoded by ORF3 of the cytoplasmic feast plasmid pGKL2 functions as a plasmid-specific capping enzyme, Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements inve stigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements. Copyright (C) 2001 John Wi ley Br Sons, Ltd.