Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p 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 chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 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 important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast 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 investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.
Tiggemann
M
M
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany.
Jeske
S
S
Larsen
M
M
Meinhardt
F
F
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D006157:Guanosine Monophosphate / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D005656:Fungal Proteins; D012315:RNA Caps; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006157:Guanosine Monophosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase