Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA–triphosphatase activities

Abstract

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 5k-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 5k-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.

Chlorella Virus RNA Triphosphatase

“…6). These findings are concordant with the conservative mutational effects for Cet1, and they suggest that Glu 24 and Glu 26 of cvRtp1 are likely to coordinate the divalent metal directly and that cvRtp1 cannot flex its structure to bring an aspartate (with its shorter main chain to carboxylate linker) into the metal coordination sphere. Replacing Glu 165 of cvRtp1 with aspartate had no salutary effect compared with the alanine mutant, whereas introducing glutamine elicited a partial restoration of function to 10% of wild-type activity (Fig.…”

“…We also introduced conservative substitutions for Glu 26 , which had been previously shown to be essential for activity (4). A total of 24 recombinant proteins with conservative changes were produced in E. coli and purified from soluble bacterial extracts by nickel-agarose chromatography (Fig.…”

“…Although the poxvirus, ASFV, and baculovirus RNA triphosphatases do contain the conserved metal-binding glutamates (equivalent to Glu 24 , Glu 26 , and Glu 165 of cvRtp1) within two motifs widely separated in the primary structure, they lack obvious counterparts of the other catalytic motifs of cvRtp1 (defined herein and corresponding to the strands of a Cet1-like tunnel). Our inference is that the vaccinia, ASFV, and baculovirus triphosphatases either lack a topologically closed tunnel architecture or, if they retain such a fold, do so without any recog- nizable amino acid sequence similarity to the Chlorella virus and yeast enzymes.…”

“…We assume, by parsimony, that separately encoded enzymatic domains are the ancestral state and that polyfunctional catalysts of the same metabolic pathway arise via gene fusion events. Polyfunctional capping enzymes related to the poxvirus and ASFV proteins are encoded by cytoplasmic plasmids of fungi (25,26). Thus, we envision a model whereby the Chlorella virus capping system arose by acquisition of nuclear capping enzymes (likely from a fungus or another lower eukaryote), whereas the polyfunctional capping enzymes of metazoan DNA viruses originated with a cytoplasmic episome (also perhaps fungal) that had already undergone fusion of the capping enzyme genes.…”

“…1) that are required for catalysis by the fungal, microsporidian, poxvirus, and baculovirus RNA triphosphatases. During our initial characterization of cvRtp1, we showed that alanine substitution of Glu 26 in the ␤1 motif (VELEFRLG) abrogated the phosphohydrolase activity (4).…”